Cell separation devices, systems, and methods

ABSTRACT

Disclosed herein are cell separation devices, methods and systems, as well as compositions and reagents for use in cell separation methods.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/272,533 filed on Dec. 29, 2015, which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE DISCLOSURE

Recent clinical trials establish that certain rare stem, progenitor orimmune cell populations have clinical utility in regenerative medicineand immunotherapy (references). Such rare target cells may be as much asfive orders of magnitude (10⁵) less numerous than the other cells inblood or bone marrow, the most numerous of which are red blood cells andplatelets. The admixture of abundant unwanted cells with rare,clinically important target cells presents a challenge in enriching andpurifying the target cells. The major cell populations in blood and bonemarrow differ in size and density, so that stratifying cells based ontheir densities during centrifugation can be utilized in the selectionand purification process. For example, normal human blood generallycomprises red blood cells (“RBCs”), the most numerous and most densecell; platelets (“PLTs”), the smallest and least dense cell; white bloodcells (“WBCs”), the largest cells, with a density between the RBCs andthe PLTs, and plasma. These three cell fractions separate into distinctpopulations during centrifugation. On average, RBCs make upapproximately 99.9% of an individual's total blood cells andapproximately 45% of the total volume of blood within an individual,although this is known to vary among individuals and, over time, withinthe same individual. RBCs serve a vital function as the principal meansof delivering oxygen to body tissues, but are not useful forregenerating tissue or enhancing the immune system to combat certaindiseases, including cancers. Nearly all of the remainder of anindividual's blood volume is made up of plasma, a non-cellular liquidaccounting for approximately 55% of the total blood volume and in whichall blood cells are suspended. Thus, over 99% of the volume of normalblood is made up of plasma and RBCs.

The remaining approximately <0.6% of the volume of normal blood consistsof WBCs and Platelets (PLTs). PLTs are small, irregularly shapedanuclear cells that outnumber the WBCs by a factor of ˜30 times. PLTsplay a fundamental role in hemostasis and healing by stopping bleedingand releasing a multitude of growth factors that repair and regeneratedamaged tissue. However, their adhesive nature interferes with theefficient enrichment and purification of the rare, clinically importanttarget cells. The least prevalent blood cells are WBCs, making up onlyabout one tenth of one percent of the total cells in a typical bloodsample. WBCs are critical to the body's immune system, and participatein the defense of the body against infectious disease, foreign materialsand hematologic and solid tumor cancers. Nearly all of the cells thatare utilized clinically in immunotherapy or regenerative medicine residewithin the WBC fraction. WBCs may be further divided into subgroups. Thelargest and most dense subgroup is the granulocytes (GRNs), which makeup approximately 60% of all WBCs. The smaller and less dense subgroupare the mononuclear cells (MNCs), which constitute the remainingapproximately 40%. MNCs can further be broken down into lymphocytes andmonocytes, but they are collectively referred to as MNCs due to thepresence in each cell of a single round nucleus. MNCs are criticalelements of the immune system, comprising T cells, B cells and NK cellsthat migrate to sites of infection in body tissue and then divide anddifferentiate into macrophages and dendritic cells to elicit an immuneresponse. Many cell therapies now being explored in clinical trialsutilize cells that reside within the MNC fraction.

Thus, in order to purify a rare population of cells from blood, bonemarrow or leukapheresis, an initial bulk depletion step to removesubstantially all of the much more numerous RBCs, GRNs and PLTs tocreate an enriched MNC fraction is desirable. As previously mentioned,this can be accomplished using centrifugation. Centrifugation is amethod of cell processing classified by the FDA as “minimallymanipulated”, which provides a simpler regulatory path for FDAclearance. Performing this initial bulk depletion process alone on bloodcan enrich the rare cell populations residing in the MNC fraction bythree orders of magnitude (10³), which makes subsequent additionalpurification and enrichment of the target cells more efficient.

Current methods for isolating target cells begins with a manual methodof isolating MNCs require a highly skilled operator working with densitygradient mediums, such as Ficoll. Density gradient mediums are smallparticles of a precise density intermediate to, for instance, thedensity of granulocytes and MNCs so that when combined and centrifuged,the particles stratify and interpose their layer in between thegranulocyte layer and the MNC layer making the subsequent pipetteretrieval of the MNCs without the presence of granulocytes moreattainable. Subsequent purification to the final target cells within theMNCs requires expensive and complex instrumentation and expensivereagents. These current methods also have low rates of throughput thatare unsuitable for purifying rare cells admixed with large quantities ofunwanted cells, or have low efficiency of target cell isolation andharvest. These methods may also expose the cells to chemical agents thatmay have undesirable effects on the cells, or require the use offunctionally open systems that present the risk of microbiologicalcontamination of the cells. Therefore, new technologies are needed toenable the isolation, separation, purification, or exchange of medium inwhich rare cells are suspended with high efficiency, high throughput,and little or no manual intervention, while employing simple equipment,cell-compatible reagents, and functionally closed, sterile systems.

SUMMARY OF THE INVENTION

The present invention provides an apparatus and method for enrichingand/or purifying essentially any or all the desired cells residing in ahost liquid, including rare desired cells such as stem, progenitor orimmune cells, while maintaining viability of the cells under asepticconditions. The practice of this invention on volumes of cell typesexemplified by blood, bone marrow or leukapheresis, which containclinically relevant numbers of stem, progenitor or immune cells, canprovide compositions of target cells that are significantly higher inrecovery, viability and purity than can be obtained by all other knowndevices.

Thus, in a first aspect, the invention provides a cell separationsystem, comprising:

(a) a cartridge comprising

-   -   (i) a processing container comprising at least one input port, a        first exit port, and a second exit port;    -   (ii) a second container comprising an input port;    -   (iii) a third container comprising an input port and a first        exit port;    -   (iv) a first conduit connecting the first exit port of the        processing container and the input port of the second container,        wherein the first conduit comprises a first reversible closing        device, wherein the second container is transiently fluidically        connected to the processing container such that fluid flow only        from the processing container to the second container may occur        when the first reversible closing device is opened;    -   (v) a second conduit connecting the second exit port of the        processing container and the input port of the third container,        wherein the second conduit comprises a second reversible closing        device, wherein the third container is transiently fluidically        connected to the processing container such that fluid flow only        from the processing container to the third container may occur        when the second reversible closing device is opened;

(b) a transfer container comprising at least one port;

(c) an at least third conduit connecting

-   -   (i) the first exit port of the third container to the at least        one port of the transfer container, and    -   (ii) the at least one port of the transfer container to the at        least one input port of the processing container    -   wherein the at least third conduit comprises at least a third        reversible closing device, such that (A) the third container is        transiently fluidically connected to the transfer container,        and (B) the transfer container is transiently fluidically        connected to the processing container; wherein the at least        third conduit is configured such that only one of the following        may be true        -   (I) fluid flow only from the third container to the transfer            container may occur when the at least third reversible            closing device is opened; or        -   (II) fluid flow only from the transfer container to the            processing container may occur when the at least third            reversible closing device is opened; and

(d) a control module configured to control activity in at least thecartridge, and the first and second conduits.

In one embodiment, the transfer container is internal to the cartridge.In another embodiment, the at least third conduit comprises a singleconduit. In another embodiment, the at least third conduit comprises Tor Y connector disposed between the third container and transfercontainer and between the transfer container and the processingcontainer.

In one embodiment, the at least one port of the transfer containercomprises a first input port and an exit port. In such an embodiment,the at least third conduit comprises:

(i) a third conduit connecting the exit port of the third container tothe input port of the transfer container, wherein the third conduitcomprises a third reversible closing device, such that the thirdcontainer is transiently fluidically connected to the transfer containersuch that fluid flow only from the third container to the transfercontainer may occur when the third reversible closing device is opened;and

(ii) a fourth conduit connecting the exit port of the transfer containerto the at least one input port of the processing container, wherein thefourth conduit comprises a fourth reversible closing device, such thatthe transfer container is transiently fluidically connected to theprocessing container, such that fluid flow only from the transfercontainer to the processing container may occur when the fourthreversible closing device is opened.

In a further embodiment, the at least one input port of the processingcontainer comprises a first input port and a second input port, whereinthe at least third conduit, or the fourth conduit (when present),connects the exit port of the transfer container to the first input portof the processing container. In such an embodiment, the cell separationsystem may further comprise a first medium input conduit connecting thesecond input port of the processing container to at least one mediumreservoir, wherein the first medium input conduit comprises at least afifth reversible closing device, wherein the at least one mediumreservoir is transiently fluidically connected to the processingcontainer such that fluid flow only from the at least one mediumreservoir to the processing container may occur when the at least fifthreversible closing device is opened.

In one embodiment, the at least one port of the transfer containerfurther comprises a second input port. In such an embodiment, the cellseparation system may further comprise a second medium input conduitconnecting the second input port of the transfer container to at leastone medium reservoir, wherein the second medium input conduit comprisesat least a sixth reversible closing device, wherein the at least onemedium reservoir is transiently fluidically connected to the processingcontainer such that fluid flow only from the at least one mediumreservoir to the transfer container may occur when the at least sixthreversible closing device is opened.

In one embodiment, the cell separation system further comprises a mixer.In one particular example, the mixer comprises a static mixer. In afurther embodiment, the mixer comprises an impeller disposed on aninternal surface of a roof of the cartridge. In another embodiment, themixer comprises an impeller spaced away from an internal surface of aroof of the cartridge. In another embodiment, the mixer comprises aperistaltic pump comprising a pump conduit having a first end and asecond end, wherein the first end of the pump conduit is positioned inthe processing chamber, and wherein the second end of the pump conduitis positioned outside of the processing chamber and is connected to theat least one input port of the processing chamber. In yet anotherembodiment, the mixer comprises a mixing module comprising a bottomportion and a top portion, wherein the cartridge is configured to bepositioned in the bottom portion, and wherein the top portion isconfigured to be removably coupled to the bottom portion. In such anembodiment, the mixing module may include a rotatable component coupledto the bottom portion, such that the rotatable component is configuredto rotate the cartridge on its vertical axis by 180 degrees or by 360degrees. In another embodiment, the mixing module may be configured toincrease a temperature of the cartridge when the cartridge is positionedin the bottom portion of the mixing module.

In one embodiment, the first medium input conduit and/or the secondmedium input conduit further comprise a filter. In another embodiment,the second container comprises an exit port coupled to a first wasteconduit. In a further embodiment, the processing container furthercomprises a sterile vent coupled to a second waste conduit.

In another aspect, the invention provides cell separation methods,comprising:

(a) processing a host liquid having a volume of at least 10 mL (or,alternatively, at least 25 mL, at least 50 mL, at least 75 mL, at least100 mL, at least 200 mL) in a functionally closed system, wherein thehost liquid comprises (i) target cells, and (ii) buoyant reagents,wherein the processing comprises contacting the target cells and buoyantreagents for a time and under conditions suitable to promote attachmentof the cells to one or more of the buoyant reagents to generate attachedtarget cells;

(b) applying a vectorial force, such as centrifugation, to the hostliquid within the functionally closed system to cause the attachedtarget cells to stratify within the host liquid; and

(c) sequestering the attached target cells to an area within thefunctionally closed system.

In one embodiment, the buoyant reagents comprise manufactured buoyantreagents, wherein each manufactured buoyant reagent comprises a buoyantreagent wherein the buoyant label and a binding agent are attached toeach other to form the manufactured buoyant reagent prior to contactingthe target cells in the host liquid. In another embodiment, the buoyantreagents comprise secondary buoyant reagents that assemble within thehost liquid, wherein the method further comprises a preprocessing step,prior to step (a), wherein the preprocessing step comprises contactingthe host liquid with buoyant labels and binding agents for a time andunder conditions suitable to promote attachment of the binding agents tothe buoyant labels to produce the buoyant reagents.

In one embodiment, each buoyant label comprises

(i) each binding agent comprises (A) a primary binding agent comprisingan agent capable of binding to at least one cellular epitope on thetarget cells, (B) a first linker bound to the primary binding agent,wherein the first linker comprises a first oligonucleotide having afirst complementary region;

(ii) each buoyant label comprises a second linker bound to the buoyantlabel;

wherein the second linker comprises a second oligonucleotide having asecond complementary region, wherein the second complementary region isperfectly complementary to the first complementary region, and wherein ahybrid of the first and second complementary regions has a calculated Tmof at least 40° C.;

wherein the preprocessing step comprises contacting the host liquid withthe buoyant labels and the binding agents for a time and underconditions suitable to promote hybridization of the first and secondcomplementary regions to produce the buoyant reagents;

wherein the processing comprises contacting the target cells and thebuoyant reagents in the host liquid under conditions suitable togenerate the attached target cells; and wherein the method furthercomprises

(d) subjecting the attached target cells to a temperature of 37° C. orless within the functionally closed system after step (c) for a timesufficient to dehybridize the first complementary region and the secondcomplementary region to release the buoyant reagents from the targetcells.

In another aspect, the invention provides cell separation methods,comprising:

(a) providing a host liquid, wherein the host liquid comprises attachedtarget cells, wherein each attached target cell comprises

-   -   (i) a binding agent bound to at least one cellular epitope on a        target cell,    -   (ii) a first linker bound to the agent, wherein the first linker        comprises a first oligonucleotide having a first complementary        region;    -   (iii) a buoyant label comprising a second linker bound to the        buoyant label, wherein the second linker comprises a second        oligonucleotide having a second complementary region, wherein        the second complementary region is perfectly complementary to        the first complementary region, wherein the second complementary        region is hybridized to the first complementary region to form a        hybrid, and wherein the hybrid of the first and second        complementary regions has a calculated Tm of at least 40° C.;

(b) applying a vectorial force, such as centrifugation, to the hostliquid to cause the attached target cells to stratify within the hostliquid;

(c) sequestering the attached target cells; and

(d) subjecting the attached target cells to a temperature of 37° C. orless after step (c) for a time sufficient to dehybridize the firstcomplementary region and the second complementary region to release thebuoyant labels from the target cells.

In one embodiment, the attached target cells are generated prior to step(a) by processing steps comprising contacting target cells in the hostliquid with manufactured buoyant reagents comprising the binding agent,the first linker, and the second linker, wherein the secondcomplementary region is hybridized to the first complementary region toform the hybrid; wherein the contacting is carried out for a time andunder conditions suitable to promote attachment of the cells to one ormore of the manufactured buoyant reagents to generate the attachedtarget cells. In another embodiment, the attached target cells aregenerated prior to step (a) by processing steps comprising: (A)contacting the host liquid with the buoyant labels and binding agentsbound to the first linker for a time and under conditions suitable topromote hybridization of the first and second complementary regions toproduce buoyant reagents; and (B) contacting the target cells and thebuoyant reagents in the host liquid for a time and under conditionssuitable to generate the attached target cells.

In either of these aspects the calculated Tm may be the Tm as calculatedusing the nearest-neighbor two-state model:

${{Tm}\left( {{^\circ}\; C} \right)} = {\frac{\Delta\; H\;{^\circ}}{{\Delta\; S\;{^\circ}} + {R\;{\ln\lbrack{oligo}\rbrack}}} - 273.15}$where ΔH° (enthalpy) and ΔS° (entropy) are the melting parameterscalculated from the sequence and the published nearest neighborthermodynamic parameters and under the ionic conditions used, R is theideal gas constant (1.987 calK⁻¹mole⁻¹), [oligo] is the molarconcentration of an oligonucleotide, and the constant of −273.15converts temperature from Kelvin to degrees of Celsius. In oneembodiment, the calculated Tm of the hybrid between the firstcomplementary region and the second complementary region is between 40°C. and about 60° C.

In another embodiment of any of the methods of the invention, thebinding agents bind to a cellular epitope on the target cells. Inanother embodiment, each buoyant reagent comprises one or more secondlinkers, wherein the one or more second linkers are bound to one or morefirst linkers attached to at least one binding agent, wherein the atleast one binding agent is capable binding to a cellular epitope on thetarget cell; and wherein the contacting comprises contacting the targetcells and the buoyant reagents for a time and under conditions suitableto promote attachment of the target cells to one or more of the buoyantreagents to generate the attached target cells. In another embodiment,the processing may comprise (i) contacting the host liquid with primarybinding agents, wherein each primary binding agent comprises (A) anagent capable of binding to at least one cellular epitope on the targetcells, and (B) a first linker bound to the agent; wherein the contactingoccurs under conditions suitable to promote attachment of the targetcells to the primary binding agents to produce target cell-binding agentcomplexes; and (ii) incubating the target cell-binding agent complexeswith the buoyant labels, wherein each buoyant label comprises a secondlinker, wherein the second linker is capable of binding to the firstlinker; wherein the incubating occurs under conditions suitable topromote binding of the first linker to the second linker to generate theattached target cells. In one embodiment, no intermediate step ofremoving unbound primary binding agents occurs between steps (i) and(ii).

In a further embodiment, the methods of the invention may furthercomprise detaching the buoyant label from the target cells within thefunctionally closed system to produce detached target cells. In oneembodiment of the methods of the invention, the target cells are thedesired cells, and the methods may comprise further steps ofconcentrating the desired cells after sequestration and producing thedetached target cells.

In another embodiment of the methods of the invention, the target cellsand/or the desired cells may be selected from the group consisting oftumor cells, cancer stem cells, hematopoietic stem and progenitor cells,mesenchymal stem and progenitor cells, adipose-derived stem andprogenitor cells, endothelial progenitor cells found in normal blood,placental/cord blood, bone marrow, white blood cells, granulocytes,mononuclear cells, lymphocytes, monocytes, T-cells, B-cells, NK cells,the stromal vascular fraction cells resident in adipose tissue, culturedcells, genetically modified cells, and sub-populations of such targetcells. In a specific embodiment, the target cells and/or the desiredcells may be selected from the group consisting of CD3+ cells, CD4+cells, CD235a, CD14+, CD19+, CD56+, CD34+, CD117⁺, KDR⁺, SIRPA⁺, ASGR1⁺,OCLN⁺, GLUT2⁺, SLC6A1⁺, TRA-1-60⁻, SSEA4⁻, AP⁻ (alkaline phosphatase),SSEA3⁻, TDGF1⁻, or CD349⁻ cells.

In various further embodiments of the methods of the invention, thetarget cells and/or the desired cells represent less than 10%, 5%, 4%,3%, 2%, or 1% of the cells in non-depleted host liquid. In variousfurther embodiments, a recovery efficiency of the desired cells isgreater than 68%, or greater than 75%, or greater than 80%, or greaterthan 85%, or greater than 90%, or greater than 95%. In otherembodiments, viability of the isolated desired cells is greater than90%, or greater than 95%, or greater than 97%, or greater than 99%. Infurther embodiments, the target cells and/or desired cells are presentat less than 20%, 10%, 5%, 4%, 3%, 2%, or 1% of total cells in the hostliquid.

In one embodiment of the methods of the invention, the binding agentsmay be selected from the group consisting of antibodies,oligonucleotides, aptamers, molecularly imprinted polymers,carbohydrates, proteins, peptides, enzymes, small molecules, lipids,fatty acids, metal atoms, metal ions or synthetic polymers. In aspecific embodiment, the binding agents comprise antibodies. In otherembodiments, the first linker and second linkers may comprise biotin,avidin, streptavidin, oligonucleotides, antibody-binding proteins,and/or moieties bound by an antibody-binding protein or any secondattached binding agent. In specific embodiments, the first linker andsecond linkers comprise biotin and/or streptavidin, either alone orcombined with oligonucleotides.

In another embodiment of the methods of the invention, the buoyantlabels may be selected from the group consisting of gas-filled bubbles,hollow polymers, glass beads, microporous beads with entrained gas,droplets of an immiscible liquid, gold nanoparticles, and silvernanoparticles. In a specific embodiment, the buoyant labels comprisegas-filled bubbles. In a further specific embodiment, the gas-filledbubbles comprise perfluorocarbon gas cores encompassed by lipid,phospholipid, carbohydrate or protein shells. In a further specificembodiment, the gas-filled bubbles comprise perfluorocarbon gas coresencompassed by a phospholipid shell. In another specific embodiment, thegas-filled bubbles may have a diameter between about 1 um and about 6.5um. In a further specific embodiment, the host liquid may be peripheralblood, cord blood, or leukapheresis.

In one embodiment, the functionally closed system for use in the methodsof the invention comprises a cell separation system comprising

(a) a cartridge comprising

-   -   (i) a processing container comprising at least one input port, a        first exit port, and a second exit port;    -   (ii) two or more additional containers, comprising at least a        second container comprising an input port; and a third container        comprising an input port and a first exit port;    -   (ii) a first conduit connecting the first exit port of the        processing container and the input port of the second container,        wherein the first conduit comprises a first reversible closing        device, wherein the second container is transiently fluidically        connected to the processing container such that fluid flow only        from the processing container to the second container may occur        when the first reversible closing device is opened;    -   (v) a second conduit connecting the second exit port of the        processing container and the input port of the third container,        wherein the second conduit comprises a second reversible closing        device, wherein the third container is transiently fluidically        connected to the processing container such that fluid flow only        from the processing container to the third container may occur        when the second reversible closing device is opened;

(b) a transfer container comprising at least one port;

(c) an at least third conduit connecting

-   -   (i) the first exit port of the third container to the at least        one port of the transfer container, and    -   (ii) the at least one port of the transfer container to the at        least one input port of the processing container    -   wherein the at least third conduit comprises at least a third        reversible closing device, such that (A) the third container is        transiently fluidically connected to the transfer container,        and (B) the transfer container is transiently fluidically        connected to the processing container; wherein the at least        third conduit is configured such that only one of the following        may be true        -   (I) fluid flow only from the third container to the transfer            container may occur when the at least third reversible            closing device is opened; or        -   (II) fluid flow only from the transfer container to the            processing container may occur when the at least third            reversible closing device is opened.

In another embodiment of the methods of the invention, the host liquidto be processed is a depleted host liquid, and prior to the processingor the preprocessing steps, the method comprises applying a vectorialforce, such as centrifugation, to non-depleted host liquid within thefunctionally closed system, such as within the processing container, todeplete non-desired cells, such as by passing the non-desired cells intothe second container, thus producing the depleted host liquid to beprocessed. In a further embodiment, the depleted host liquid is passedfrom the processing container to the transfer container and mixed withthe buoyant reagents to initiate processing the host liquid. In oneembodiment, the target cells are the desired cells, and wherein thesequestering comprises passing the detached target cells to the thirdcontainer. In another embodiment, the target cells are not the desiredcells, and the sequestering comprises concentrating the detached targetcells within the functionally closed system. In a further embodiment,the cell separation system further comprises a control module forcontrolling the activity in at least the cartridge and the first andsecond conduits. In one embodiment of any of the methods of theinvention, the functionally closed system may comprises the cellseparation system of any embodiment or combination of embodiments of theinvention.

In another aspect, the invention provides cell suspensions, comprising

(a) a liquid medium having a volume of at least 1 mL (or, alternatively,at least 2 ml, 5 ml, 10 ml, 15 ml, 30 ml, 25 mL, at least 50 mL, atleast 75 mL, at least 100 mL, at least 200 mL); and

(b) desired cells suspended in the liquid medium, wherein the desiredcells are selected from the group consisting of hematopoietic stem andprogenitor cells, mesenchymal stem and progenitor cells, endothelialprogenitor cells found in normal blood, placental/cord blood, bonemarrow, white blood cells, granulocytes, mononuclear cells, lymphocytes,monocytes, T-cells, B-cells, NK cells, the stromal vascular fractioncells resident in adipose tissue, cultured cells, genetically modifiedcells, and sub-populations of such desired cells, wherein the desiredcells are present in a liquid medium and wherein the desired cells makeup at least 80% of cells in the cell suspension;

wherein the desired cell viability is greater than 90%, or greater than95%, or greater than 97%, or greater than 99%; and

wherein the cell suspension is present within a functionally closed cellseparation system, or is directly obtained from the functionally closedcell separation system without further processing.

In one specific embodiment, the desired cells may be selected from thegroup consisting of CD3+ cells, CD4+ cells, CD235a, CD14+, CD19+, CD56+,CD34+, CD117⁺, KDR⁺, SIRPA⁺, ASGR1⁺, OCLN⁺, GLUT2⁺, SLC6A1⁺, TRA-1-60⁻,SSEA4⁻, AP⁻ (alkaline phosphatase), SSEA3⁻, TDGF1⁻, or CD349⁻ cells. Inanother embodiment, the functionally closed cell separation systemcomprises the cell separation system of any embodiment or combination ofembodiments of the invention. In one embodiment, the cell suspension maybe present within the transfer container, the processing containerand/or the third container. In another embodiment, the cell suspensionis present in a cell suspension removal stream via the transfercontainer of the functionally closed cell separation system.

In various embodiments, the number of viable desired cells is at least1×10³, or at least 1×10⁴, or at least 2×10⁴, or at least 5×10⁴, or atleast 1×10⁵, or at least, 2×10⁵, or at least 5×10⁵, or at least 1×10⁶,or at least 2×10, or at least 5×10⁶, or at least 1×10⁷, or at least2×10⁷, or at least 5×10⁷, or at least 1×10⁸, or at least 2×10⁸, or atleast 5×10⁸, or at least 1×10⁹, or at least 2×10⁹, or at least 5×10⁹. Inanother embodiment, the desired cells comprise a buoyant label attachedto the cells.

In a further aspect, the invention provides compositions comprising:

(a) at least one binding agent covalently or non-covalently linked to atleast one first linker, the at least one binding agent able to bind toat least one molecular target on the cells in a cell suspension; and

(b) at least one buoyant label covalently or non-covalently linked to atleast one second complementary linker, the at least one buoyant labelexhibiting a density substantially different from the density of theliquid medium in which the cells to be separated are suspended.

In another aspect, the invention provides kits comprising

(i) a primary binding agent comprising an agent capable of binding to atleast one cellular epitope on a target cell;

(ii) a first linker bound to the agent, wherein the first linkercomprises a first oligonucleotide having a first complementary region;

(iii) a buoyant label;

(iv) a second linker bound to the buoyant label, wherein the secondlinker comprises a second oligonucleotide having a second complementaryregion, wherein the second complementary region is perfectlycomplementarity to the first complementary region, and wherein a hybridof the first and second complementary regions has a calculated Tm of atleast 40° C.

In another aspect, the invention provides compositions comprising

(i) a primary binding agent comprising an agent capable of binding to atleast one cellular epitope on a target cell;

(ii) a first linker bound to the agent, wherein the first linkercomprises a first oligonucleotide having a first complementary region;

(iii) a buoyant label;

(iv) a second linker bound to the buoyant label, wherein the secondlinker comprises a second oligonucleotide having a second complementaryregion perfectly complementary to the first complementary region,wherein a hybrid of the first and second linkers' complementary regionshas a calculated Tm of at least 40° C.

wherein the first linker and the second linker are hybridized to eachother. In one embodiment, the composition further comprises a targetcell bound to the primary binding agent.

In one embodiment of the compositions and kits of the invention, thecalculated Tm may be the Tm as calculated using the nearest-neighbortwo-state model:

${{Tm}\left( {{^\circ}\; C} \right)} = {\frac{\Delta\; H\;{^\circ}}{{\Delta\; S\;{^\circ}} + {R\;{\ln\lbrack{oligo}\rbrack}}} - 273.15}$

where ΔH° (enthalpy) and ΔS° (entropy) are the melting parameterscalculated from the sequence and the published nearest neighborthermodynamic parameters, R is the ideal gas constant (1.987calK⁻¹mole⁻¹), [oligo] is the molar concentration of an oligonucleotide,and the constant of −273.15 converts temperature from Kelvin to degreesof Celsius. In one embodiment, the calculated Tm of the hybrid betweenthe first complementary region and the second complementary region isbetween 40° C. and about 60° C.

In various embodiments of the kits and compositions of the invention,the primary binding agent may be selected from the group consisting ofantibodies, oligonucleotides, aptamers, molecularly imprinted polymers,carbohydrates, proteins, peptides, enzymes, small molecules, lipids,fatty acids, metal atoms, metal ions or synthetic polymers. In onespecific embodiment, the primary binding agent comprises an antibody. Inanother specific embodiment, one of the first linker and the secondlinker further comprises biotin (optionally linked to a first member ofan oligonucleotide hybridizing pair), and the other further comprisesstreptavidin (optionally linked to a second member of an oligonucleotidehybridizing pair). In various further embodiments, the buoyant labelsmay be selected from the group consisting of gas-filled bubbles, hollowpolymers, glass beads, microporous based with entrained gas, droplets ofan immiscible liquid, gold nanoparticles, and silver nanoparticles. Inone specific embodiment, the buoyant labels comprise gas-filled bubbles.In various embodiments, the gas-filled bubbles may compriseperfluorocarbon gas cores encompassed by lipid, phospholipid, protein orcarbohydrate shells. In a further specific embodiment, the gas-filledbubbles comprise perfluorocarbon gas cores encompassed by phospholipidshells. In another specific embodiment, the gas-filled bubbles have adiameter between about 1 um and about 6.5 um.

In another aspect, the invention provides compositions comprisingdesired cells purified via buoyancy-activated cell sorting from astarting admixture of at least one molecular type of desired cell and atleast one molecular type of non-desired cell where said startingadmixture contains at least 1 times the number of non-desired cells asdesired cells, or at least 5 times the number of non-desired cells asdesired cells, or at least 10 times the number of non-desired cells asdesired cells, or at least 50 times the number of non-desired cells asdesired cells, or at least 100 times the number of non-desired cells asdesired cells, or at least 500 times the number of non-desired cells asdesired cells, or at least 1000 times the number of non-desired cells asdesired cells wherein:

the recovery efficiency of the at least one type of desired cell isgreater than 80%, or greater than 85%, or greater than 90%, or greaterthan 95%;

the purity of the at least one type of desired cell is greater than 80%,or greater than 85%, or greater than 90%, or greater than 95%;

the viability of the at least one type of desired cell is greater than90%, or greater than 95%, or greater than 97%, or greater than 99%; and

the volume of the admixture of at least one molecular type of desiredcell and at least one molecular type of non-desired cell subjected tobuoyancy-activated cell sorting is greater than 10 mL, or greater than50 mL, or greater than 100 mL, or greater than 150 mL, or greater than200 mL, or greater than 400 mL, or greater than 800 mL.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an exploded view of a cell separation system, according to anexample embodiment.

FIG. 2 is a cross-section view of a cartridge of a cell separationsystem, according to an example embodiment.

FIG. 3 is a side view of a cell separation system, according to anexample embodiment.

FIG. 4A is a perspective view of an example static mixer on the roof ofthe cartridge, according to an example embodiment.

FIG. 4B is a bottom view of the example static mixer of FIG. 4A,according to an example embodiment.

FIG. 5A is a perspective view of another example static mixer on theroof of the cartridge, according to an example embodiment.

FIG. 5B is a bottom view of the example static mixer of FIG. 5A,according to an example embodiment.

FIG. 6 is a cross-section view of an example mixer in the funnel of thecartridge, according to an example embodiment.

FIG. 7 is a cross-section view of an example peristaltic pump of thecartridge, according to an example embodiment.

FIG. 8 is an exploded view of an example mixing module, according to anexample embodiment.

FIG. 9 shows an exemplary flow chart of an embodiment of the inventionin which the desired cells are CD3+ cells, and the methods compriseusing the cell separation system described herein.

FIG. 10 shows an exemplary flow chart of an embodiment of the inventionin which two or more differential centrifugation steps are carried outto sequentially remove non-desired cells.

FIG. 11 summarizes experimental data generated using exemplaryembodiments of the methods of the invention.

FIG. 12 summarizes experimental data generated using exemplaryembodiments of the methods and systems of the invention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

As used herein, the term ‘about” means+/−5% of the recited parameter.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words ‘comprise’, ‘comprising’, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”. Words using the singular or pluralnumber also include the plural and singular number, respectively.Additionally, the words “herein,” “above,” and “below” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of theapplication.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While the specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize.

With reference to the Figures, FIG. 1 illustrates an example cellseparation system 100. As shown in FIG. 1, the cell separation system100 may include a cartridge 102, a control module 104, and a dockingstation 106. As used herein, “cartridge” is a closed housing (having aroof) that allows the aseptic transfer of cells between containerswithin the closed housing and the mixing of cells, binding agents,buoyant labels, and buoyant reagents to accomplish linkage, comprisingthree or more mechanically joined containers which are transientlyfluidically connected. The cartridge 102 may hold up to 250 mL ofliquid, may be cylindrical, may be single-use, and may be constructedpreferably of hard plastic, and more preferably optically clearpolycarbonate. In certain other embodiments, the cartridge 102 isreusable. The control module 104 may be removably coupled to thecartridge 102. The control module 104 is an electro-mechanical devicewith optical and gravitational sensing. In particular, the controlmodule 104 provides optical sensing of cell interfaces in the bottom ofthe cartridge 102, and may be configured to control one or morereversible closing devices to control activity between variouscontainers of the cartridge 102, as discussed in additional detailbelow. The docking station 106 may be removably coupled to the controlmodule 104, and may be used to recharge the control module 104. Further,the docking station 106 may receive one or more protocols wirelessly orthrough a wired connection, and may further be configured to downloadand process data received by the control module 104. The docking station106 preferably uses a rechargeable battery system to power the controlmodule 104 that monitors and controls gravitational and optical sensingequipment and directs activity in the cartridge 102. The means fordetermining a G force may be any commonly known in the art, such ascalculating said force through a measurement of centrifuge RPM, orthrough direct measurement of acceleration or force.

When the cartridge 102 is removably attached to the control module 104,one or more detectors, such as optical sensors or other sensors of thecontrol module 104 may be used to detect the type of cells flowingthrough the cartridge 102. Further, the control module 104 may alsoinclude at least one but preferably two or more optical or otheremitters. In an exemplary embodiment, four infrared emitters/detectorpairs are arranged vertically in the control module 104. In a preferredembodiment, infrared sensors are located directly across from pairedinfrared emitters. In second preferred embodiment, transmitters thatprovide wavelengths that are preferentially absorbed by red cells arelocated directly across from paired sensors sensitive to that frequency.In a third preferred embodiment sensors are utilized that identify cellsthat have absorbed fluorescent dyes. In the first preferred embodiment,the presence of cells interferes with the emitted infrared light and theinfrared light detector quantifies the amplitude of the signalpenetrating the fluid. In a preferred embodiment the sensors may assignthe level of transmission a value from 0-1000. Pure plasma, whichsimilar to water blocks none of the infrared light, will register avalue of roughly 1000. As compacted RBCs pass between the sensor/emitterpairs, essentially all infrared light is blocked and the detectorregisters a value of 0.

FIG. 2 illustrates a cross-section view of the cartridge 102. As shownin FIG. 2, the cartridge 102 includes a processing container 108comprising at least one input port 110, a first exit port 112, and asecond exit port 114. The cartridge 102 also includes a second container116 comprising an input port 118, and a third container 120 comprisingan input port 122 and a first exit port 124. As described in the processbelow, a biological fluid containing cells, such as normal blood, cordblood or bone marrow, is delivered to the processing container 108through the at least one input port 110. The second container 116 of thecartridge 102 comprises a large first rigid storage compartment or RBCdepletion compartment, and the third container 120 comprises a smallersecond rigid storage compartment or SC compartment into which the WBCsand substantially all the SPCs are transferred. The second container 116is significantly larger than the third container 120, as the volume ofRBCs depleted from a blood sample is always much greater that the volumeof WBCs collected. All compartments are distinct from one another withinthe cartridge 102, but contiguous with respect to airflow. The secondcompartment 116 and the third compartment 120 may be connected by smallfiltered or sufficiently narrowed air vents to the processing container108 so as to allow displacement of air as cell solutions move from theprocessing chamber 108 into the second and third containers 116, 120,but does not permit fluid transfer between the containers. In oneembodiment, the processing container 108 is an approximately conicalcentral container, while the second and third containers 116, 120 aresmaller, circumferentially located containers. The containers may be ofany suitable volume for a given purpose. In one embodiment, the thirdand second containers 116, 120 each may further comprise additional,normally closed ports providing optional points of connection to anysuitable receiving containers external to the cartridge 102 for gravitydraining or as a result of adding air pressure through the air filter192. In a further embodiment, fluid in the processing container 108 maybe removed through the internal input tube 110 by the application of airpressure through air filter 192 with that ceases when the level of fluidin the container drops below the lowest point on the internal rigidinput tube 110.

As shown in FIG. 2, the cartridge 102 also includes a first conduit 126connecting the first exit port 112 of the processing container 108 andthe input port 118 of the second container 116. The first conduit 126comprises a first reversible closing device 128. The second container116 is transiently fluidically connected to the processing container 108such that fluid flow only from the processing container 108 to thesecond container 116 may occur when the first reversible closing device128 is opened. The cartridge 102 also includes a second conduit 130connecting the second exit port 114 of the processing container 108 andthe input port 122 of the third container 120. The second conduit 130comprises a second reversible closing device 132. The third container120 is transiently fluidically connected to the processing container 108such that fluid flow only from the processing container 108 to the thirdcontainer 120 may occur when the second reversible closing device 132 isopened.

As used herein, a “reversible closing device” is any device that can beclosed (such as a by a controller) to prohibit fluid flow. Exemplarysuch devices include, but are not limited to valves, clamps, andstopcock. As used herein, “transiently fluidically connected” means thatthe containers are fluidically non-continuous (that is, eachfunctionally closed) other than when transiently connected via openingof the device's normally closed valves to achieve aseptic transfer offluid or cell suspension from one container to another. The “conduits”may be any suitable device to permit fluid transfer between thecontainers, including but not limited to tubing. All of the conduitsbecome “normally closed”, such that the containers are not fluidicallyconnected, as soon as the operator removes a pin installed duringcartridge assembly that prevents the conduits from being closed. Theconduits may be closed by any suitable reversible closing device, suchas a valve, clamp or stopcock. In one embodiment, the conduits withinthe cartridge may be closed by a spring loaded, tube pinching mechanismat all times except when fluids should pass, at which time the pinchingmechanism may be rotated (for example, by a control module automaticallycontrolling the reversible opening of the conduit) to allow passage ofthe fluids, and then may be rotated again to allow the spring loadedtube pinching mechanism to close off that passage by re-pinching theconduit. In such an example, the reversible closing devices comprise twoopposing clamps having pinching surfaces approximately 0.088 incheswide, and require approximately 1.6 pounds of pinching force to blockall fluid passage through a urethane tube with an inner diameter of0.062 inches and an exterior diameter of 0.088 inches when the hydraulicpressure in the tube is at 325 PSI. Pinching forces in excess of 1.6pounds may be required at greater pressures, and reduced pinching forcesmay be sufficient at lower pressures. In such an example, a cantileversystem may be used to achieve these required pinching pressures. Thecantilever system may open and close the conduits (pinch and release thetubing) as needed. Springs may be provided on each cantilever, and arepreferably located at the extreme end of the cantilever. The actuatorovercomes the resistance of the springs to move the lever. Once theactuator stops applying force, the bias of the springs urges the leverback to its first position.

As described above, the processing container 108, or other containersmay optionally be interrogated by at least one detector for detectingthe presence or absence of cells. In such an embodiment, the controlmodule 104 controls opening and closing of the first reversible closingdevice 128 and/or the second reversible closing device 132 based oninformation relayed from the at least one detector. Any suitabledetector may be used, including but not limited to optical detectors, asdiscussed above in relation to FIG. 1.

As will be described in detail below, in operation, the RBCs initiallymigrate towards the bottom of the processing container 108, movingradially outward away from the axis of rotation of the centrifuge untilreaching the bottom of the processing container 108, where the firstreversible closing device 128 and the second reversible closing device132 reside. Here, the pressure head of fluid above the bottom of theprocessing container 108 urges the fluid into one of two compartments:either the second container 116 or the third container 120. Whichcompartment the fluid is directed into is dependent upon the status(open, closed) of the first reversible closing device 128 and the secondreversible closing device 132. In either case, after passing through thefirst reversible closing device 128 and/or the second reversible closingdevice 132, the fluid flows generally toward the axis of rotation, urgedby pressure from the of fluid (mostly plasma) remaining in theprocessing container 108. The fluid that has passed through the firstreversible closing device 128 and/or the second reversible closingdevice 132 is then retained in either the second container 116 or thethird container 120. Through minute adjustments of the first reversibleclosing device 128 and/or the second reversible closing device 132,unwanted cell solutions may be depleted and desired cell solutions maybe harvested.

As shown in FIG. 3, the cell separation system 100 may further include atransfer container 134 comprising at least one port 136. The transfercontainer 134 may be mechanically coupled to the cartridge 102, or maybe physically connected only via the relevant conduit connecting thetransfer container 134 to containers within the cartridge 102 tomaintain the system as a functionally closed system. As such, in oneembodiment, the transfer container 134 is internal to the cartridge 102;in another embodiment the transfer container 134 is external to thecartridge 102. The various containers may comprise any suitablematerial, such as a rigid structure, a bag, a bottle, or any othersuitable structure. In one embodiment, each container is a rigidcontainer, such as a hard plastic container (including but not limitedto polycarbonate). In another embodiment, the transfer container 134 isa flexible container, including but not limited to a bag. The transfercontainer 134 can be a rigid container, because the air in the containerat the start of the transfer of harvested target cells in solution candisplace along the relevant conduit connecting it to the processingcontainer. The transfer container 134 also can be a flexible container,which has the advantage of being foldable into a small shape to make iteasier to store, for example, in the rotor compartment of a centrifugeduring centrifugation.

Further, as shown in FIG. 3, the cell separation system 100 furtherincludes an at least third conduit 138 connecting the first exit port124 of the third container 120 to the at least one port 136 of thetransfer container 134, and the at least one port 136 of the transfercontainer 134 to the at least one input port 110 of the processingcontainer 108. The at least third conduit 138 comprises at least a thirdreversible closing device 140, such that the third container 120 istransiently fluidically connected to the transfer container 134, and thetransfer container 134 is transiently fluidically connected to theprocessing container 108. The at least third conduit 138 is configuredsuch that only one of the following may be true: (I) fluid flow onlyfrom the third container 120 to the transfer container 134 may occurwhen the at least third reversible closing device 140 is opened, or (II)fluid flow only from the transfer container 134 to the processingcontainer 108 may occur when the at least third reversible closingdevice 140 is opened.

The third conduit 138 between the first exit port 124 of the thirdcontainer 120 to the transfer container 134 and from the transfercontainer 134 to the at least one input 110 of the processing container108 is also normally closed by any suitable reversible closing device.In one embodiment, the third conduit 138 may be clamped on the exteriorof the cartridge 102 by the third reversible closing device 140 (justadjacent to the first exit port 124 of the third container 120) and maybe unclamped at the time of transfer of sequestered attached cells fromthe third container 120 to the transfer container 134 (for example, bygravity draining the sequestered attached cells from the third container120 to the transfer container 134). Following transfer into the transfercontainer 134, the third reversible closing device 140 on the thirdconduit 138 adjacent to the third container port 124 may be re-clamped.At that time, for example, the sequestered attached cells may betransferred to the processing container 108 via the third conduit 138(for example, by gravity draining the sequestered attached cells backinto the processing chamber 108, for further processing—such asisolating/enriching a sub-population of the sequestered, attached cells,including but not limited to mononuclear cells to join the cell-free andplatelet free plasma from the host liquid). The transfer container 134therefore provides an economic advantage as the cartridge 102 may bereused. Additionally, the transfer container 134 allows for improvedmixing the target cell suspension and BACS reagents.

The cell separation system 100 may further include a control module 104,as discussed above in relation to FIG. 1. The control module 104 may beconfigured to control activity in at least the cartridge 102, and thefirst and second conduits 126, 130. In some embodiments, the controlmodule 104 may also control activity within the transfer container 134and/or within the third conduit 138. For example, the control module 104may control activity within the transfer container 134 and/or within thethird conduit 138 in embodiments in which the transfer container 134 ispresent within the cartridge 102. The control module 104 may control thereversible closing devices 128, 132, 140 that direct the flow of fluidbetween the cartridge containers 108, 116, 120, such as when placed in acentrifuge. In one non-limiting embodiment, the cartridge 102 containingthe target cell/buoyant label mixture is centrifuged so that targetcells that bind to the buoyant label separate from the cells not boundto the buoyant label. The control module 104 may be programmed todeliver the non-buoyant pelleted cells to the second container 116 ofthe cartridge 102 via the first reversible closing device 128, leavingthe bulk of the supernatant and substantially all of the target cellsthat bound to the buoyant reagent in the processing container 108 of thecartridge 102.

In one embodiment, the at least third conduit 138 comprises a singleconduit. In this embodiment, the only a single conduit is fluidicallycoupled to the transfer container 134, and the conduit can befluidically separated such that fluid flowing from the third container120 to the transfer container 134 is separated from fluid flowing fromthe transfer container 134 to the processing container 108. Any suitablereversible closing means can be used in this embodiment. Onenon-limiting example is shown in FIG. 3, in which the at least thirdconduit 138 comprises a T or Y connector 144 disposed between the thirdcontainer 120 and transfer container 134, and between the transfercontainer 134 and the processing container 108, with appropriatereversible closing devices 140, 146 to regulate the desired fluid flow.

In a further embodiment, the at least one port of the transfer containercomprises a first input port 148 and an exit port 150. In thisembodiment, the at least third conduit 138 may comprise (i) a thirdconduit 138 connecting the exit port 124 of the third container 120 tothe input port 136 of the transfer container 134, wherein the thirdconduit 138 comprises a third reversible closing device 140, such thatthe third container 120 is transiently fluidically connected to thetransfer container 134 such that fluid flow only from the thirdcontainer 120 to the transfer container 134 may occur when the thirdreversible closing device 140 is opened, and (ii) a fourth conduit 152connecting the exit port 136 of the transfer container 134 to the atleast one input port 110 of the processing container 108, wherein thefourth conduit 152 comprises a fourth reversible closing device 146,such that the transfer container 134 is transiently fluidicallyconnected to the processing container 108, such that fluid flow onlyfrom the transfer container 134 to the processing container 108 mayoccur when the fourth reversible closing device 146 is opened. In thisembodiment, separation of fluid flowing from the third container 120 tothe transfer container 134 away from fluid flowing from the transfercontainer 134 to the processing container 108 is made possible by theuse of completely separate conduits 138, 152.

In a still further embodiment, the at least one input port 110 of theprocessing container 108 comprises a first input port 154 and a secondinput port 156, wherein the at least third conduit 138, or the fourthconduit 152 (when present), connects the exit port 136 of the transfercontainer 134 to the first input port 154 of the processing container108. In such an example, the cell separation system 100 may furthercomprise a first medium input conduit 158 connecting the second inputport 156 of the processing container 108 to at least one mediumreservoir 160, wherein the first medium input conduit 158 comprises atleast a fifth reversible closing device 162, wherein the at least onemedium reservoir 160 is transiently fluidically connected to theprocessing container 108 such that fluid flow only from the at least onemedium reservoir 160 to the processing container 108 may occur when theat least fifth reversible closing device 162 is opened.

The medium input reservoir(s) 160 can be used to supply host liquid,binding agents, buoyant labels, and/or buoyant reagents to theprocessing container 108 via the second input port 156.

In yet another embodiment, the at least one input port 110 of theprocessing container 108 comprises a third input port 157. The thirdinput port 157 may be coupled to a fifth conduit 159. In such anexample, liquid can be removed from the processing chamber 108 byproviding positive pressure at the filter 186.

In one embodiment, the at least one port of the transfer container 134further comprises a second input port 161. For example, the cellseparation system 100 may further comprise a second medium input conduit163 connecting the second input port 161 of the transfer container 134to at least one medium reservoir (not shown), wherein the second mediuminput conduit 163 comprises at least a sixth reversible closing device165, wherein the at least one medium reservoir is transientlyfluidically connected to the processing container 108 such that fluidflow only from the at least one medium reservoir to the transfercontainer 134 may occur when the at least sixth reversible closingdevice 165 is opened.

The media input reservoir(s) can be used to supply host liquid, bindingagents, buoyant labels, and/or buoyant reagents to the transfercontainer 134 via the second input port 161. In embodiments where atleast one medium reservoir is transiently fluidically connected to boththe processing container 108 and the transfer container 134, the mediareservoir(s) may be the same reservoirs, or each container may have itsown dedicated reservoir(s).

In a further embodiment, the cell separation system 100 furthercomprises a mixer. The mixer may be used, for example, topromote/improve mixing of (a) the buoyant reagents and the host liquid,and/or (b) the buoyant labels, binding agents, and linkers (whenpresent), and/or (c) buoyant labels, binding agents, linkers (whenpresent), and host liquid. In one embodiment, the mixer is a staticmixer. A static mixer comprises container with an input and exit thatremains stationary while providing continuous mixing of two fluidssimultaneously passing through the container, in a configuration suchas, but not limited to, a cylindrical tube containing mixer elementsoriented throughout the interior length of the cylindrical tube.

Exemplary static mixers appropriate for mixing in the devices of theinvention (such as mixing a target cell suspension (major component) andBACS reagents (additive)) are shown in FIGS. 4A-5B. The mediumreservoirs described above may be transiently fluidically connected tothe static mixer via the fifth and sixth reversible closing devices. Inone non-limiting embodiment, as shown in FIGS. 4A and 4B, the staticmixer 164 acts to rotate the cartridge 102 on its axis by including animpeller 166 affixed to the internal surface 168 of the cartridge roof170 causing, for example, a BACS reagent and target cells in solutionwithin the processing container to admix. The advantage of the design inFIGS. 4A-4B is that the mixing addition to the interior of the cartridgeis glued in place, so mixing of the in cell solution and the BACSreagents occurs merely by placing the loaded cartridge on a roller tabledevice for a programmable time.

In another non-limiting embodiment, as shown in FIGS. 5A and 5B, thestatic mixer 164 acts to rotate the cartridge 102 on its axis byincluding an impeller 166 spaced away from the internal surface 168 ofthe cartridge roof 170 causing, for example, a BACS reagent and targetcells in solution within the processing container to admix. Theadvantage of the design in FIGS. 5A-5B is that the mixing addition tothe interior of the cartridge rotates on its axis, driven by a motormeans, so that the cartridge remains upright and does not have to beremoved from the centrifuge.

In another non-limiting embodiment, as shown in FIG. 6, the mixer actsto impart circulating motion to an admixture of BACS reagent and targetcells in solution within the processing container 108 of the cartridge102 while the cartridge 102 remains motionless. Such an embodiment mayinclude an impeller 172 positioned within a cylindrical tube 174, with amotor 176 configured to drive the impeller 172 to thereby cause themixing in the processing container 108.

In another non-limiting embodiment, as shown in FIG. 7, the mixercomprises a peristaltic pump 178 comprising a pump conduit 180 having afirst end 182 and a second end 184, wherein the first end 182 of thepump conduit 180 is positioned in the processing chamber 108, andwherein the second end 184 of the pump conduit 180 is positioned outsideof the processing chamber 108 and is connected to the at least one inputport 110 of the processing chamber 108.

In yet another non-limiting embodiment, as shown in FIG. 8, the mixercomprises a mixing module 194. The mixing module may include a bottomportion 195 and a top portion 196. As shown in FIG. 8, the cartridge 102may be configured to be positioned in the bottom portion 195, and thetop portion 196 may be configured to be removably coupled to the bottomportion 195. As such, the cartridge 102 may be positioned in a chambercreated by the bottom portion 195 and the top portion 196. In onenon-limiting embodiment, the mixing module 194 may include a drive shaft197 coupled to the bottom portion 195. The drive shaft 197 may beconfigured to rotate the cartridge 102 on its vertical axis. The driveshaft 197 may be coupled to a motor, which in turn causes the bottomportion 195 of the mixing module 194 to rotate. In one particularexample, the drive shaft 197 is configured to rotate the cartridge 102on its vertical axis by 180 degrees, so the bottom of the cartridge 102is in a vertical position, and then the drive shaft 197 causes thecartridge 102 to rotate back 180 degrees to an original uprightposition. In another example, the drive shaft 197 is configured torotate the cartridge 102 end over end continuously over 360 degrees atvarious rotational rates for a period of time. In another non-limitingembodiment, the bottom portion 195 of the mixing module 194 may beconfigured to vibrate to assist in mixing. In another non-limitingembodiment, the mixing module 194 may be configured to increase atemperature of the cartridge 102 when the cartridge is positioned 102 inthe bottom portion 195 of the mixing module 194. Such an increase intemperature may occur through conduction, convection, or radiationheating in the bottom portion 195 of the mixing module 194.

As shown in FIG. 8, the mixing module 194 may include a mixing controlmodule 198, which may include a control panel 199 and a display 200. Thecontrol panel 199 may be used to select a time period for mixing, aswell as one or more mixing parameters. For example, a user could select180 degree mixing for a period of time, 360 degree mixing for a periodof time, heating at a specific temperature for a period of time, and/orvibrating for a period of time. Other examples are possible as well.

In various embodiments, the first medium input conduit 158 and/or thesecond medium input conduit further comprise a filter 186. Any suitablefilter (including but not limited to a 0.2 micron filter, or otherappropriately sized filter to remove large particles) can be used inconnection with the medium input conduit(s) to promote asepticintroduction of medium into processing container and/or targetcontainer. Exemplary placement of such filters can be seen, for example,in FIG. 3.

In another embodiment, the second container 116 comprises an exit port188 coupled to a first waste conduit 190. This embodiment permitsremoval of waste product from the second container 116. In a furtherembodiment, the processing container 108 further comprises a sterilevent 192 coupled to a second waste conduit (not shown). This embodimentpermits removal of waste product from the processing container 108.Exemplary placement of such a sterile vent 192 can be seen, for example,in FIGS. 1 and 2.

In another aspect, the invention provides methods for cell separation,comprising:

(a) processing a host liquid having a volume of at least 10 mL (or,alternatively, at least 25 mL, at least 50 mL, at least 75 mL, at least100 mL, at least 200 mL, etc.) in a functionally closed system, whereinthe host liquid comprises (i) target cells, and (ii) buoyant reagents,wherein the processing comprises contacting the target cells and buoyantreagents for a time and under conditions suitable to promote attachmentof the cells to one or more of the buoyant reagents to generate attachedtarget cells,

(b) applying a vectorial force, such as centrifugation, to the hostliquid within the functionally closed system to cause the attachedtarget cells to stratify within the host liquid; and

(c) sequestering the attached target cells to an area within thefunctionally closed system.

All steps in the methods of this aspect of the invention are carried outin the functionally closed system to maintain sterility and to permitthe processing of much larger volumes of host liquid for isolation ofdesired cells with greater efficiency and viability, and at higherconcentration than was possible using previous cell separation methods.All fluid transfer and processing steps occur under aseptic conditionsand the environment remains closed and sterile.

As used herein, a “functionally closed system” is a container orcollection of containers which remain(s) internally aseptic whilepermitting intermittent fluid transfers between aseptic containersand/or gas exchange with its surrounding atmosphere.

As summarized in Table 1 below, processing of cord blood and peripheralblood (exemplary host liquids) enables depletion of red blood cells(RBCs) by 99.6%, granulocytes by 80% and platelets by 84%, to producemononuclear cells (MNC) enriched fractions (referred to in the table as“BACS MNC prep,” to the left of the bold vertical line running throughthe table). Following this initial MNC enrichment, RBCs and plateletsstill predominate, such that MNCs still represent only a small fractionof the enriched cell population, and specific cells expressing anextracellular molecular target, such as CD34+ and CD3+ cells representonly a very low percentage of the enriched MNC cell preparation. Thepresent invention provides a dramatic increase in the ability to isolatetarget cells harboring a molecular marker of interest, by furtherdepleting RBC to a final depletion of 99.999%, granulocytes 99.9999%(cord blood) or 99.96% (peripheral blood) and platelets 98.2% (referredto in the table as “BACS final” and ‘BACS cell selection efficiency”, tothe right of the bold vertical line running through the table). Themethods of the invention enable depletion of non-target MNCs to 99.993%(cord blood) or 98.788% (peripheral blood) whilst recovering targetcells with a selection efficiency of 79% (cord blood) or 80% (peripheralblood). The methods of the invention thus provide an exponentialincrease in the ability to isolate target cells of interest in a cellpopulation from volumes of starting host liquid that are necessary forclinical applications. The present invention's combination of very highretention of target cells in the preliminary MNC preparation step andhigh recovery of target cells in the BACS cell isolation step, resultingin high overall efficiency, illustrates the commercial and therapeuticbenefits of the present invention in the frequently encounteredsituations where either cell manufacturing costs or therapeutic outcomesare optimized by using as many target cells as possible.

As shown in the examples that follow, the methods of the presentinvention provide satisfactory target cell recovery efficiencies(greater than 70%) at cell densities ranging from just 5×10⁶ cells permL to at least 105×10⁶ cells per mL—more than a 20-fold range, and thelatter a suspension in which cells comprise roughly 20% of the totalvolume. This robust and consistent performance over such a wide celldensity range, including at very high cell densities, is a surprisingresult because the buoyant labels themselves are large particles(um-scale nominal diameters, such as gas-filled bubbles)) withaccordingly limited ability to translate through dense suspensions,unlike much smaller reagents such as magnetic nanoparticles (nominaldiameter 50 nm). In order to achieve high recovery efficiencies everycell likely needs to bind to multiple buoyant reagents. This, combinedwith the likelihood that not every cell/bubble collision will result ina productive binding event, renders it surprising that relatively largebuoyant reagents can be mixed with a very dense cell suspensionsufficiently well to achieve satisfactory target cell recoveries inreasonable times, without the application of shear forces that wouldunacceptably compromise cell viability.

As used herein, a “host liquid” is a raw source of desired cells(including but not limited to whole blood, placental/cord blood, bonemarrow, leukapheresis, buffy coat, a suspension of cultured cells, or ofgenetically modified cells, or of other manufactured cells, etc.),admixed with non-desired cells, which may be diluted or undiluted (forexample, diluted with buffers or any other liquid useful in isolatingdesired cells, such as saline, phosphate buffered saline, a cell culturemedium, a protease solution, etc.). In one embodiment, the host liquidis peripheral blood, cord blood, or leukapheresis, or diluted versionsthereof.

In one embodiment, the host liquid may be a “non-depleted” host liquid,in that all cells normally present in the relevant host liquid remainpresent. In another embodiment, the host liquid may comprise a depletedhost liquid. A “depleted” host liquid is a host liquid from which asubstantial fraction (for example, at least 50%; in other embodiments,at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more) of one or more types of non-desired cells havebeen depleted. By way of non-limiting examples, a depleted host liquidmay comprise desired cells in suspension after depletion of red bloodcells from whole blood via density centrifugation, differentialcentrifugation, or lysis, or from which red blood cells and granulocyteshave been depleted, or from which platelets have been depleted, or maycomprise a suspension of genetically modified cells from whichnon-modified cells have been depleted, or may comprise a digested tissuesample from which non-stem cells have been depleted, or may comprise adigested tumor sample from which non-neoplastic cells have beendepleted. In all embodiments of depleted host liquid, the desired cellsmay be suspended in any suitable liquid, including but not limited tosaline, phosphate buffered saline, a cell culture medium, diluted orundiluted original host liquid (i.e.: whole blood, etc.).

As used herein, a “desired cell” (plural, “desired cells”) is the cellor cells to be enriched or isolated in an intermediate output or thefinal output of the methods of this invention.

As used herein, a “target cell” (plural, “target cells”) is a cell orcells to which buoyant reagents are attached in order to separate themfrom other cells by the methods of the present invention. In oneembodiment, the target cells are the desired cells (i.e.: positiveselection). In another embodiment, the target cells make up all or aportion of the non-desired cells (i.e.: negative selection). The methodsand systems of the invention may be used with any suitable target cell.In various non-limiting embodiments, the target cells may be selectedfrom the group consisting of tumor cells, cancer stem cells,hematopoietic stem and progenitor cells, mesenchymal stem and progenitorcells, adipose-derived stem and progenitor cells, endothelial progenitorcells found in normal blood, placental/cord blood, bone marrow, whiteblood cells, granulocytes, mononuclear cells, lymphocytes, monocytes,T-cells, B-cells, NK cells, the stromal vascular fraction cells residentin adipose tissue, cultured cells, genetically modified cells, andsub-populations of such target cells. In various specific embodiments,the target cells may be of CD3+ cells, CD4+ cells, CD235a, CD14+, CD19+,CD56+, CD34+, CD117⁺, KDR⁺, SIRPA⁺, ASGR1⁺, OCLN⁺, GLUT2⁺, SLC6A1⁺,TRA-1-60⁻, SSEA4⁻, AP⁻ (alkaline phosphatase), SSEA3⁻, TDGF1⁻, or CD349⁻cells cells.

In one embodiment, the host liquid comprises a dilution of an initialcell suspension of desired cells. In this embodiment, the host liquidimmediately before processing by the methods of this invention may bediluted up to 50×, to 40×, 30×, 20×, 10×, 4× (i.e.: 4-fold), up to 3×,up to 2×, up to 1×, up to 0.5×, up to 0.25×, up to 0.1×, or less asappropriate for a given cell separation procedure. Any suitable diluentmay be used. In one non-limiting embodiment, the desired cells are CD3+cells, and the host liquid may comprise a dilution of between 2×-4× ofthe initial undiluted host liquid.

As used herein a “buoyant label” is a material that (upon cellattachment) results in a complex of cell and buoyant labels with adensity substantially different from the density of target cell aloneand/or the density of the host liquid. Suitable such buoyant labels mayinclude, without limitation, gas-encapsulating bubbles with protein orlipid shells, hollow polymers, glass beads (either hollow or solid),microporous beads with entrained gas, droplets of an immiscible liquid,gold nanoparticles, and silver nanoparticles. In one specificembodiment, the buoyant labels comprise gas-encapsulating bubbles, suchas those encompassed by protein, lipid, phospholipid, or carbohydrateshells. In one embodiment, the gas-filled bubbles compriseperfluorocarbon gas cores encompassed by a phospholipid shell. In any ofthese embodiments, the gas-filled bubbles may have any suitablediameter; in one non-limiting embodiment, the gas-filled bubbles have adiameter between about 1 um and about 6.5 um.

In one embodiment, the buoyant label comprises at least one bindingagent attached to it, either covalently or non-covalently; theattachment may optionally be via a linker. This embodiment is referredto herein as a “buoyant reagent”. As used herein, a “binding agent” is astructure, such as a molecule, that is capable of binding withsufficiently high affinity and specificity to at least one cellularepitope on at least one target cell. Suitable binding agents mayinclude, without limitation, antibodies, oligonucleotides, aptamers,molecularly imprinted polymers, carbohydrates, proteins, peptides,enzymes, small molecules, lipids, fatty acids, metal atoms, metal ionsand synthetic polymers. In one embodiment, the binding agent comprisesan antibody that selectively binds to a cellular epitope on the targetcell. As used herein, a “primary binding agent” is a binding agent thathas no buoyant label attached to it. As used herein, a “linker” (or,individually “a linker”) comprises a pair of chemical moieties (a firstlinker and a second linker) attached covalently or non-covalently one toa binding agent the other to a buoyant label, which are able tospontaneously attach (either covalently or non-covalently) to each otherin a suitable medium under suitable conditions with sufficiently highaffinity to achieve the indirect connection of a binding agent to abuoyant label, via the linkers, to form a buoyant reagent. In certainembodiments, the at least one first linker moieties are functionallyexposed avidin or streptavidin, and the second linker moieties arebiotin or a biotin derivative, either alone or linked witholigonucleotide binding pairs as described herein.

In other embodiments, the first linker is a first oligonucleotide andthe second linker is a second oligonucleotide complementary over atleast a portion of its length (such as fully complementary) to the firstoligonucleotide and capable of binding to the first oligonucleotide viabase pairing.

In certain embodiments, a combination of two or more different buoyantlabels are employed, one or more of these having a density substantiallygreater than the density of the liquid medium and one or more having adensity substantially less than the liquid medium. The one or morebinding agents attached to the one or more different buoyant labels maytarget one or more different molecular targets or may all target thesame molecular target, and the one or more molecular targets may be onthe desired cells or the undesired cells.

In certain embodiments, the quantity of the one or more binding agentsadded to the host liquid in binding agent-first order is sufficient tosubstantially saturate the binding agent's binding sites on the targetcells of the cell suspension while leaving only an amount of unboundbinding agent remaining in the mixture that is insufficient tosubstantially interfere with the binding of buoyant label to thecell-bound binding agent, thus obviating the need to remove unboundbinding agent prior to adding buoyant label.

In further embodiments, the quantity of the one or more binding agentsto add to the host liquid is determined by a preceding count of thenumber of target cells present in the host liquid, employing anysuitable means known to those skilled in the art, for example, withoutlimitation a hematology analyzer, flow cytometry, microscopy,sedimentation, enzymatic assay, ELISA, or the like. In certainembodiments, the quantity of the one or more binding agents to add tothe host liquid is determined by testing a range of two or morequantities of binding agents against aliquots of a host liquid. Infurther embodiments, the quantity of the one or more binding agents usedis up to 40 times the number of binding agent binding sites present onthe target cells.

The various binding agents, linkers, and buoyant labels may becollectively referred to as “buoyancy-activated cell sorting reagents”(BACS reagents).

The buoyant reagents may be produced by any suitable combination of thebuoyant labels and binding agents/optional linkers including, but notlimited to, manufactured buoyant reagents, secondary buoyant reagents,binding agent-first order buoyant reagents, buoyant-first order buoyantreagents, and simultaneous buoyant reagents. As used herein, a“manufactured buoyant reagent” is a buoyant reagent wherein the buoyantlabel and the binding agent are attached to each other to form a buoyantreagent prior to contacting the host liquid.

As used herein, a “secondary buoyant reagent” is a buoyant reagent,which spontaneously assembles when host liquid is contacted with abuoyant label and a separate binding agent. Thus, in some embodiments,the buoyant reagents comprise secondary buoyant reagents that assemblewithin the host liquid, wherein prior to processing the host liquid togenerate attached target cells, the method further comprisespreprocessing steps comprising contacting the host liquid with buoyantlabels and binding agents for a time and under conditions suitable topromote attachment of the binding agents to the buoyant labels toproduce the buoyant reagents. Any suitable order of addition of thebuoyant label and separate binding agent to the host liquid can beemployed as suitable for an intended purpose. As used herein, “bindingagent-first order” is an order of forming a secondary buoyant reagent bycontacting the host liquid first with the binding agent, and thensubsequently contacting the cell suspension with the buoyant label. Asused herein, “buoyant-first order” is an order of forming a secondarybuoyant reagent by contacting the host liquid first with the buoyantlabel, and then subsequently contacting the host liquid with the bindingagent. As used herein, “simultaneous order” is an order of forming asecondary buoyant reagent by contacting the host liquid substantiallysimultaneously with both the buoyant label and the binding agent.

In one non-limiting embodiment,

(i) each binding agent comprises (A) a primary binding agent comprisingan agent capable of binding to at least one cellular epitope on thetarget cells, (B) a first linker bound to the primary binding agent,wherein the first linker comprises a first oligonucleotide having afirst complementary region; and

(ii) each buoyant label comprises a second linker bound to the buoyantlabel;

wherein the second linker comprises a second oligonucleotide having asecond complementary region, wherein the second complementary region isperfectly complementary to the first complementary region, and wherein ahybrid of the first and second complementary regions has a calculated Tmof at least 40° C.;

wherein the preprocessing step comprises contacting the host liquid withthe buoyant labels and the binding agents for a time and underconditions suitable to promote hybridization of the first and secondcomplementary regions to produce the buoyant reagents;

wherein the processing comprises contacting the target cells and thebuoyant reagents in the host liquid under conditions suitable togenerate the attached target cells; and wherein the method furthercomprises:

(d) subjecting the attached target cells to a temperature of 37° C. orless within the functionally closed system after step (c) for a timesufficient to dehybridize the first complementary region and the secondcomplementary region to release the buoyant reagents from the targetcells.

In another aspect, the invention provides cell separation methods,comprising:

(a) providing a host liquid, wherein the host liquid comprises attachedtarget cells, wherein each attached target cell comprises

-   -   (i) a binding agent bound to at least one cellular epitope on a        target cell,    -   (ii) a first linker bound to the agent, wherein the first linker        comprises a first oligonucleotide having a first complementary        region;    -   (iii) a buoyant label comprising a second linker bound to the        buoyant label, wherein the second linker comprises a second        oligonucleotide having a second complementary region, wherein        the second complementary region is perfectly complementary to        the first complementary region, wherein the second complementary        region is hybridized to the first complementary region to form a        hybrid, and wherein the hybrid of the first and second        complementary regions has a calculated Tm of at least 40° C.;

(b) applying a vectorial force, such as centrifugation, to the hostliquid to cause the attached target cells to stratify within the hostliquid;

(c) sequestering the attached target cells; and

(d) subjecting the attached target cells to a temperature of 37° C. orless after step (c) for a time sufficient to dehybridize the firstcomplementary region and the second complementary region to release thebuoyant labels from the target cells.

In this aspect, the methods may be carried out in a functionally closedsystem, or may be carried out in an open system. The methods may becarried out in any suitable volume. In one embodiment of this aspect,the methods are carried out in volumes of at least 1 ml, 2 ml, 5 ml, 10ml, 25 ml, 50 ml, 100 ml, or at least 200 ml.

In one embodiment, the attached target cells are generated prior to step(a) by processing steps comprising contacting target cells in the hostliquid with manufactured buoyant reagents comprising the binding agent,the first linker, and the second linker, wherein the secondcomplementary region is hybridized to the first complementary region toform the hybrid; wherein the contacting is carried out for a time andunder conditions suitable to promote attachment of the cells to one ormore of the manufactured buoyant reagents to generate the attachedtarget cells. In another embodiment, the attached target cells aregenerated prior to step (a) by processing steps comprising:

(A) contacting the host liquid with the buoyant labels and bindingagents bound to the first linker for a time and under conditionssuitable to promote hybridization of the first and second complementaryregions to produce buoyant reagents; and

(B) contacting the target cells and the buoyant reagents in the hostliquid for a time and under conditions suitable to generate the attachedtarget cells

In these different embodiments/aspects, the first oligonucleotidecomprises a first complementary region that is perfectly complementaryto a second complementary region in the second oligonucleotide. As willbe understood by those of skill in the art, the first and secondoligonucleotides may comprise additional nucleotides as suitable for anintended purpose (for example, to bind the oligonucleotide to anothercomponent of the linker, such as biotin or streptavidin). In thisembodiment, the hybridized complementary oligonucleotides link bindingagents (such as antibodies) to the buoyant label (such as gas filledbubbles) in a reversible fashion, enabling the release of target cellsfrom their bound bubbles by raising the temperature of the cellsuspension sufficiently to dehybridize the hybrid formed between thefirst and second complementary regions. The inventors have surprisinglydiscovered that hybrids with a theoretical Tm of 40° C. or greater canbe used in the methods of the invention, even though such hightemperatures, if they were required to achieve detachment of bubblesfrom isolated target cells, would be expected to compromise theviability of most mammalian cell types (which prefer a maximum of 37°C.). Surprisingly, the inventors discovered that the hybrids with atheoretical Tm of 40° C. or greater effectively releases bubbles fromcells at well below the calculated Tm, i.e.: at about 37° C. While notbeing bound by any mechanism, the inventors believe that release wellbelow the theoretical Tm is due to the large Brownian, centripetal,and/or frictional forces imposed on these hybrids by themicrometer-diameter cells and bubbles which they tether together, whichmay weaken the hybrids below their theoretical Tm. As a consequence theinventors have routinely achieved 97% to 99% target cell viability inthese embodiments, with recovery efficiency of up to 90%.

In one embodiment, the hybridization step can be carried out attemperatures ranging from 4° C. to about 30° C. In various furtherembodiments, the hybridization step can be carried out at temperaturesranging from 10° C. to about 30° C., 15° C. to about 30° C., 20° C. toabout 30° C., 21° C. to about 30° C., 22° C. to about 30° C., 23° C. toabout 30° C., 24° C. to about 30° C., 25° C. to about 30° C., 10° C. toabout 25° C., 15° C. to about 25° C., 20° C. to about 25° C., 21° C. toabout 25° C., 22° C. to about 25° C., 23° C. to about 25° C., or 24° C.to about 25° C.

In these embodiments, the calculated Tm is the Tm as calculated usingthe nearest-neighbor two-state model:

${{Tm}\left( {{^\circ}\; C} \right)} = {\frac{\Delta\; H\;{^\circ}}{{\Delta\; S\;{^\circ}} + {R\;{\ln\lbrack{oligo}\rbrack}}} - 273.15}$where ΔH° (enthalpy) and ΔS° (entropy) are the melting parameterscalculated from the sequence and the published nearest neighborthermodynamic parameters under the ionic conditions used, R is the idealgas constant (1.987 calK⁻¹mole⁻¹), [oligo] is the molar concentration ofan oligonucleotide, and the constant of −273.15 converts temperaturefrom Kelvin to degrees of Celsius. The nearest neighbor thermodynamicparameters are those of Allawi, H., Santa Lucia, J. Jr., Biochemistry,36, 10581, and the monovalent cation correction is that of Owczarzy, R.et al., Biochemistry, 47, 5336

In various embodiments, the calculated Tm of the hybrid between thefirst complementary region and the second complementary region isbetween 40° C. and about 60° C., between 40° C. and about 58° C., 40° C.and about 56° C., 40° C. and about 55° C., 40° C. and about 54° C., 40°C. and about 53° C., 40° C. and about 52° C., 40° C. and about 51° C.,40° C. and about 50° C., 41° C. and about 60° C., between 41° C. andabout 58° C., 41° C. and about 56° C., 41° C. and about 55° C., 41° C.and about 54° C., 41° C. and about 53° C., 41° C. and about 52° C., 41°C. and about 51° C., 41° C. and about 50° C., 42° C. and about 60° C.,between 42° C. and about 58° C., 42° C. and about 56° C., 42° C. andabout 55° C., 44° C. and about 54° C., 42° C. and about 53° C., 42° C.and about 52° C., 42° C. and about 51° C., or 42° C. and about 50° C.

As will be understood by those of skill in the art, the specificnucleotide sequence of the first and second complementary regions may beany that forms perfect complements over the length of the first andsecond complementary regions, resulting in a hybrid having a calculatedTm as recited.

A “vectorial force” is a force having a direction as well as amagnitude, including but not limited to gravitational force, centripetalforce, and centrifugal force.

Those of skill in the art are well aware of the relative densities ofthe main cellular components of blood, the Cluster of Differentiation(CD) surface markers on specific cell types and the relationship ofthose cell types to various medical treatment indications.

The methods of the invention can be carried out in any suitable,functionally closed cell separation system. In one non-limitingembodiment, the cell separation system may comprise the system describedin U.S. Pat. No. 8,747,289. In an improved embodiment, the cellseparation device comprises the functionally closed cell separationsystem of any embodiment or combination of embodiments of the systemsdisclosed herein. In one such embodiment, the functionally closed systemcomprises a cell separation system comprising

(a) a cartridge comprising

-   -   (i) a processing container comprising at least one input port, a        first exit port, and a second exit port;    -   (ii) two or more additional containers, comprising at least a        second container comprising an input port; and a third container        comprising an input port and a first exit port;    -   (ii) a first conduit connecting the first exit port of the        processing container and the input port of the second container,        wherein the first conduit comprises a first reversible closing        device, wherein the second container is transiently fluidically        connected to the processing container such that fluid flow only        from the processing container to the second container may occur        when the first reversible closing device is opened;    -   (v) a second conduit connecting the second exit port of the        processing container and the input port of the third container,        wherein the second conduit comprises a second reversible closing        device, wherein the third container is transiently fluidically        connected to the processing container such that fluid flow only        from the processing container to the third container may occur        when the second reversible closing device is opened;

(b) a transfer container comprising at least one port;

(c) an at least third conduit connecting

-   -   (i) the first exit port of the third container to the at least        one port of the transfer container, and    -   (ii) the at least one port of the transfer container to the at        least one inlet port of the processing container    -   wherein the at least third conduit comprises at least a third        reversible closing device, such that (A) the third container is        transiently fluidically connected to the transfer container,        and (B) the transfer container is transiently fluidically        connected to the processing container; wherein the at least        third conduit is configured such that only one of the following        may be true        -   (I) fluid flow only from the third container to the transfer            container may occur when the at least third reversible            closing device is opened; or        -   (II) fluid flow only from the transfer container to the            processing container may occur when the at least third            reversible closing device is opened.

As used herein, a “reversible closing device” is any device that can beclosed (such as a by a controller) to prohibit fluid flow. Exemplarysuch devices include, but are not limited to valves, clamps, andstopcock.

As used herein, “cartridge” is a closed housing (having a roof) thatallows the aseptic transfer of cells between containers within theclosed housing and the mixing of cells, binding agents, buoyant labels,and buoyant reagents to accomplish linkage, comprising three or moremechanically joined containers which are transiently fluidicallyconnected. As used herein, “transiently fluidically connected” meansthat the containers are fluidically non-continuous (that is, eachfunctionally closed) other than when transiently connected via openingof the device's normally closed valves to achieve aseptic transfer offluid or cell suspension from one container to another. The “transfercontainer” is transiently fluidically coupled to the third container viathe second conduit, and transiently fluidically coupled to theprocessing container, but there is no requirement that the transfercontainer be mechanically coupled to the cartridge, except via therelevant conduit.

The “conduits” may be any suitable device to permit fluid transferbetween the containers, including but not limited to tubing. All of theconduits are “normally closed”, such that the containers are notfluidically connected. The conduits may be closed by any suitablereversible closing device, such as a valve, clamp or stopcock. In oneembodiment, the conduits within the cartridge may be closed by a springloaded, tube pinching mechanism at all times except when fluids shouldpass, at which time the pinching mechanism may be rotated (for example,by a control module automatically controlling the reversible opening ofthe conduit) to allow passage of the fluids, and then may be rotatedagain to close off that passage by re-pinching the conduit.

The third conduit between the exit of the third container to thetransfer container and from the transfer container to the input of theprocessing container is also normally closed by any suitable reversibleclosing device. In one embodiment, the third conduit may be clamped onthe exterior of the cartridge (just adjacent to the exit port of thethird container) and may be unclamped at the time of transfer ofsequestered attached cells from the third container to the transfercontainer (for example, by gravity draining the sequestered attachedcells from the third container to the transfer container). Followingtransfer into the transfer container, the clamp on the third conduitadjacent to the third container port may be reestablished. At that time,for example, the sequestered attached cells may be transferred to theprocessing container via the third conduit (for example, by gravitydraining the sequestered attached cells back into the processingchamber, for further processing—such as isolating/enriching asub-population of the sequestered, attached cells, including but notlimited to mononuclear cells to join the cell-free and platelet freeplasma from the host liquid).

The transfer container may be mechanically coupled to the cartridge, ormay be physically connected only via the relevant conduit connecting thetransfer container to containers within the cartridge to maintain thesystem as a functionally closed system.

The various containers may comprise any suitable material, such as arigid structure, a bag, a bottle, or any other suitable structure. Inone embodiment, each container is a rigid container, such as a hardplastic container (including but not limited to polycarbonate). Inanother embodiment, the transfer container is a flexible container,including but not limited to a bag. The transfer container can be arigid container, because the air in the container at the start of thetransfer of harvested target cells in solution can displace along therelevant conduit connecting it to the processing container. The transfercontainer also can be a flexible container, which has the advantage ofbeing foldable into a small shape to make it easier to store, forexample, in the rotor compartment of a centrifuge during centrifugation.

In one embodiment, the processing container is an approximately conicalcentral container, while the second and third containers are smaller,circumferentially located containers. The containers may be of anysuitable volume for a given purpose.

In one embodiment, the third and second containers each may furthercomprise additional, normally closed ports providing optional points ofconnection to any suitable receiving containers external to thecartridge. In another embodiment, the processing, second, and thirdcontainers may share a common filtered air vent to provide airdisplacement as fluids move in and out of all the containers and thetransfer container of the cartridge. In the normal operation of thecartridge this filtered air vent does not permit fluid transfer betweenthe containers. FIG. 9 shows an exemplary flow chart of an embodiment ofthe invention in which the desired cells are CD3+ cells, and the methodscomprise using the cell separation system described herein.

In another embodiment, the methods comprise contacting the target cellsand buoyant labels for a time and under conditions suitable to promoteattachment of the target cells to one or more of the buoyant labels togenerate attached target cells. In one embodiment, each buoyant reagentcomprises one or more second linkers, wherein the one or more secondlinkers are bound to one or more first linkers attached to at least onebinding agent, wherein the at least one binding agent is capable bindingto a cellular epitope on the target cell; and wherein the contactingcomprises contacting the target cells and the buoyant reagents for atime and under conditions suitable to promote attachment of the targetcells to one or more of the buoyant reagents to generate the attachedtarget cells.

In this embodiment, the processing comprises generation of buoyantreagents within the functionally closed system; this can be done in thepresence or absence of the target cells. In non-limiting embodiments,generation of buoyant reagents may be carried out by mixing:

(A) (i) firstly at least one binding agent capable of binding to atleast one molecular target and bearing at least one first linker; (ii)secondly at least one buoyant label bearing at least one complementarylinker; or

(B) (i) firstly at least one buoyant label bearing at least one linker;(ii) secondly at least one binding agent capable of binding to the atleast one molecular target and bearing at least one complementarylinker; or

(C) at least one buoyant label bearing at least one linker and at leastone binding agent capable of binding to the at least one moleculartarget and bearing at least one complementary linker substantiallysimultaneously.

In another embodiment, the methods comprise generating targetcell-binding agent complexes first, as follows:

(i) contacting the host liquid with primary binding agents, wherein eachprimary binding agent comprises (A) an agent capable of binding to atleast one cellular epitope on the target cells, and (B) a first linkerbound to the agent; wherein the contacting occurs under conditionssuitable to promote attachment of the target cells to the primarybinding agents to produce target cell-binding agent complexes; and

(ii) incubating the target cell-binding agent complexes with the buoyantlabels, wherein each buoyant label comprises a second linker, whereinthe second linker is capable of binding to the first linker; wherein theincubating occurs under conditions suitable to promote binding of thefirst linker to the second linker to generate the attached target cells.

In this embodiment, the method may comprise no intermediate step ofremoving unbound primary binding agents occurs between steps (i) and(ii). This embodiment may be referred to as a “no-wash” protocol. Asshown in the examples that follow, the no-wash protocol routinelyyielded excellent target cell recoveries (from 70+% to 90+%) across awide variety of source materials, cells concentrations, process volumes,targets, manual or automated protocols, and buoyant reagent (such asgas-filled bubble) diameters. The successful application of the no-washprotocol across many source materials and process conditions is asurprising result. Workers normally skilled in the art of physicalcapture of target cells using binding agents (such as antibodies) andparticles (such as microbeads, nanoparticles, or microbubbles) know thatwithout detailed preliminary antibody titration studies (on eachindependent biological specimen) it is impossible to predict the totalnumber of antibody binding sites in a given target cell suspension. Inthe absence of such titration studies (which are time-consuming andconsume source material), adding a standard quantity of antibody may beexpected to sometimes result in a substantial excess of not-cell-boundantibody remaining in the cell suspension, which would be expected tocompete with cell-bound antibody for binding to particles in said secondstep, thus resulting in low target cell recovery efficiency. The no-washprotocol confers valuable benefits. Normally, in order to spare theend-user the requirement of a wash step, a manufacturer would pre-labelparticles with binding agents, such as antibodies, during manufacturing.This transfers the typically requisite wash step from the end-user tothe manufacturer, increasing manufacturing costs (and thus reagentprices). Manufacturers employing this strategy are also presented withthe requirement of assembling and stocking a large number of differentantibody-labeled microbubble products (one for each distinct targetmolecule of interest to customers). This leads to complex manufacturingprocesses and inventories of numerous distinct products, againincreasing manufacturers' costs and thus product prices. Also, in thecommon case where the buoyant reagent component of a labeled buoyantreagent product has a shorter inherent shelf lives than does the bindingagent component, this also leads to wastage of expensive binding agentsas inventory expires, again increasing costs.

In another embodiment, a manufactured buoyant reagent capable of bindingto the at least one molecular marker can be used. In each of theseembodiments, the mixing of buoyant reagents and target cells can becarried out in the transfer container, the processing container, orboth.

In one embodiment, the buoyant reagents are generated in the transfercontainer. In other embodiments, the buoyant reagents may be generatedin the processing chamber, or in an external mixing device.

In a further embodiment, contacting the buoyant reagents with the targetcells to produce the attached target cells is carried out in thetransfer container.

Mixing of the BACS regents and target cells in solution can beaccomplished via any suitable means that accounts for the fact that thebuoyant labels rise while target cells will fall in the host liquid. Inone embodiment, the mixing is carried out so that substantially everybinding site on every target cell is bound with a buoyant label, toimprove efficiency. In one non-limiting embodiment, mixing is carriedout by providing nutating motion to the BACS reagent and the solution oftarget cells in the processing container of the cartridge, such as byplacing a device of the invention on a rotating or shaking platform. Ina further embodiment, the mixing comprises periodic re-orienting of thecartridge by 180 degrees to reverse the intrinsic rising motion of thebuoyant label and lowering motion of the target cells to move toward theopposite interior surfaces of the processing container.

In another non-limiting embodiment, mixing is carried out by providingnutating motion to the BACS reagent and the target cells in solution inthe transfer container. In a further embodiment, the mixing comprisesperiodic re-orienting of the transfer container by 180 degrees to causethe intrinsic rising motion of the buoyant label and falling motion ofthe target cells to move toward the opposite interior surfaces of thetransfer container.

In further non-limiting embodiments, the mixing is carried out by usinga cell separation system comprising an on-board mixer. Embodiments ofsuch on-board mixers are described below, in further disclosure relatingto the cell separation system.

In another embodiment, the host liquid to be processed is a depletedhost liquid, and prior to the processing or the preprocessing steps, themethod comprises applying a vectorial force, such as centrifugation, tonon-depleted host liquid within the functionally closed system, such aswithin the processing container, to deplete non-desired cells, such asby passing the non-desired cells into the second container, thusproducing the depleted host liquid to be processed. This embodimentpermits the steps of depletion of non-desired cells and subsequentpurification of target/desired cells to be carried out in the samefunctionally closed system, which was not possible using prior artmethods. In the two-step process, the initial step of enrichmentincreases the relative abundance of cells of interest versus cells notof interest, to levels sufficiently greater than that of the startingmaterial to permit subsequent processing to proceed efficiently. In aspecific embodiment this enrichment step is performed via differentialcentrifugation in any embodiment or combination of embodiments of thesystems described herein. In the second step, the output cell suspensionof the initial (enrichment) step is subjected to the processing stepsusing the buoyant reagents to achieve recovery of as many target cellsas possible, of sufficient purity for their intended application (i.e.,contaminated by as few not-of-interest cells as required). In thetwo-step process, it is often the case that some cells of interest arelost during the initial enrichment step. Hence there is here also anoverall recovery metric: percentage of target cells present in thestarting material that are recovered in the output of the BACS step.

As will be understood by those of skill in the art, the application ofthe vectorial force may be carried out a single time to removenon-desired cells from the host liquid, or two or more differentialcentrifugation steps may be carried out to sequentially removenon-desired cells. An example of this process can be seen in FIG. 10.

As will be understood by those of skill in the art, producing thedepleted host liquid may comprise application of a single round ofapplying vectorial force, such as centrifugation, to the non-depletedhost liquid within the functionally closed system, such as within theprocessing container, to deplete non-desired cells.

Alternatively, depleting the host liquid may comprise two or more roundsof applying the vectorial force (such as centrifugation) to removedifferent types of non-desired cells to produce the depleted host liquidto be further processed to isolate the desired target cells (FIG. 10).For example, when the host liquid is whole blood, a first round ofcentrifugation may be used to deplete, for example, red blood cellsonly, or red blood cells and granulocytes, with a second round ofcentrifugation to stratify mononuclear cells and platelets to form thedepleted host liquid.

In one non-limiting embodiment, the functionally closed system canreceive a host liquid (such as normal blood) that comprises a source ofdesired cells, into the processing container and, when placed in acentrifuge, will perform an initial desired cell enrichment step duringcentrifugation, including transfer of non-desired cells (such as RBCs,GRNs and PLTs) to the second container within the cartridge and provideaseptic transfer of the enriched desired cell preparation (such as MNCs)including the rare desired cells from the third container and then backinto the processing container for subsequent further purification viabinding to the buoyant labels and isolation as described above. Thenon-desired cells such as RBCs, GRNs, PLTs and excess fluid (such asplasma) can be removed from the functionally closed system withoutdisturbing the enriched target cells, all while the system remainsfunctionally closed.

In one embodiment, the depleted host liquid is passed from theprocessing container to the transfer container and mixed with thebuoyant reagents to initiate processing step (a) to produce the attachedtarget cells. In a further embodiment, the method further comprisesdetaching the buoyant label from the target cells within thefunctionally closed system to produce detached target cells. Anysuitable method to detach the buoyant label from the target cells can beused including, but not limited to, disruption of the buoyant reagents(for example, via the application of positive or negative pressure orultrasonic energy that will not damage the cells), or by breaking thelinkages between the binding agents bound to the target cells and thebuoyant labels those binding agents are linked to (for example viadehybridization of oligonucleotide linkers or enzymatic cleavage ofmacromolecular linkers or chemical cleavage of small-molecule linkers),thus releasing the target cells to, for example, migrate down theconical processing container of the cartridge (now substantially free ofnon-target cells) during centrifugation, to be positioned for collectioninto the cartridge's third container. In certain embodiments, the devicefor applying said energy or pressure (such as a sonicator) is integralto the functionally closed cell separation system. In certain otherembodiments, the device for applying said energy or pressure is integralto one of the containers of the system. In certain embodiments, saidenergy or pressure is separate from any container of the system. Theinventors have surprisingly routinely obtained high target cellviabilities (90% to 100%), coupled with high target cell recoveryefficiency, using sonication for detaching the target cells from thebuoyant reagents.

In certain embodiments, the method is employed to achieve positiveselection. In this embodiment, the target cells are the desired cells,and the method further comprises concentrating the detached target cellswithin the functionally closed system, wherein the sequesteringcomprises passing the concentrated detached target cells to the thirdcontainer. In other embodiments, the method is employed to achievenegative selection (i.e.: the target cells are not the desired cells).In this embodiment, the sequestering comprises concentrating thedetached target cells within the functionally closed system. In certainother embodiments, two or more rounds of negative and/or positiveselection are employed sequentially. In certain embodiments, employingtwo or more sequential rounds of selection, cells are directed fromeither the second or third container back to the processing container,with or without mixing of the directed cells with additional BACSreagents.

In another embodiment, the cell separation system further comprises acontrol module for controlling the activity in at least the cartridgeand the first and second conduits.

In some embodiments, the controller may also control activity within thetransfer container and/or within the third conduit. For example, thecontroller may control activity within the transfer container and/orwithin the third conduit in embodiments in which the transfer containeris present within the cartridge. The controller may control thereversible closing devices that direct the flow of fluid between thecartridge containers, such as during centrifugation. In one non-limitingembodiment, the cartridge containing the target cell/buoyant labelmixture is centrifuged so that target cells that bind to the buoyantlabel separate from the cells not bound to the buoyant label. Thecontrol module may be programmed to deliver the non-buoyant pelletedcells to the second container of the cartridge via the first reversibleclosing device, leaving the bulk of the supernatant and substantiallyall of the target cells that bound to the buoyant reagent in theprocessing container of the cartridge.

In a further embodiment, the processing container or other containersmay optionally be interrogated by at least one detector for detectingthe presence or absence of cells. In this embodiment, the control modulecontrols opening and closing of the reversible closing devices within atleast the first and/or second conduits based on information relayed fromthe at least one detector. Any suitable detector may be used, includingbut not limited to optical detectors.

In all embodiments described herein, the desired cells may be any cellsof interest, including but not limited to hematopoietic stem andprogenitor cells, mesenchymal stem and progenitor cells, adipose-derivedstem and progenitor cells, endothelial progenitor cells found in normalblood, placental/cord blood, bone marrow, white blood cells,granulocytes, mononuclear cells, lymphocytes, monocytes, T-cells,B-cells, NK cells, the stromal vascular fraction cells resident inadipose tissue, cultured cells, genetically modified cells, andsub-populations of such target cells. In various non-limitingembodiments, the desired cells may be CD3+ cells, CD4+ cells, CD235a,CD14+, CD19+, CD56+, CD34+, CD117⁺, KDR⁺, SIRPA⁺, ASGR1⁺, OCLN⁺, GLUT2⁺,SLC6A1⁺, TRA-1-60⁻, SSEA4⁻, AP⁻ (alkaline phosphatase), SSEA3⁻, TDGF1⁻,or CD349⁻ cells. This list includes negative markers (indicated by asuperscript minus sign), where the methods are used to purify cellsnegative for the recited marker. The negative markers provided in thislist are pluripotency markers, and thus depleting cells having one ormore of these negative markers can be used to deplete differentiatedcells of residual pluripotent cells (i.e., iPSCs or ESCs).

In various embodiments, the host liquid has a volume of at least 10 ml(or, alternatively, at least 25 mL, at least 50 mL, at least 75 mL, atleast 100 mL, at least 200 mL, etc.).

In other embodiments, the desired cells represent less than 10% (or,alternatively, less than 5%, 1%, 0.5%, 0.1%, 0.05%, etc.) of the cellsin the non-depleted host liquid. In one non-limiting embodiment, CD3+,CD4+, CD14+, CD19+, CD56+, or CD34+ cells are the desired cells andrepresent less than about 0.2% in the non-depleted host liquid (wholeblood), and represent than about 20%, 18%, 15%, 12%, 10%, 5%, 2.5%, 1%,or 0.5% of cells in depleted host liquid (such as whole blood after redblood cell and/or platelet depletion)

In still further embodiments, a recovery efficiency of the desired cellsis greater than 68%, or greater than 75%, or greater than 80%, orgreater than 85%, or greater than 90%, or greater than 95%. In onenon-limiting embodiment, CD3+ cells, CD4+ cells, CD235a, CD14+, CD19+,CD56+, CD34+, CD117⁺, KDR⁺, SIRPA⁺, ASGR1⁺, OCLN⁺, GLUT2⁺, SLC6A1⁺,TRA-1-60⁻, SSEA4⁻, AP⁻ (alkaline phosphatase), SSEA3⁻, TDGF1⁻, or CD349⁻cells are the desired cells and have recoveries greater than 68%, orgreater than 75%, 80%, 85%, or 90%.

In other embodiments, viability of the desired cells is greater than90%, or greater than 95%, or greater than 97%, or greater than 99%. Inone non-limiting embodiment, CD3+ cells, CD4+ cells, CD235a, CD14+,CD19+, CD56+, CD34+, CD117⁺, KDR⁺, SIRPA⁺, ASGR1⁺, OCLN⁺, GLUT2⁺,SLC6A1⁺, TRA-1-60⁻, SSEA4⁻, AP⁻ (alkaline phosphatase), SSEA3⁻, TDGF1⁻,or CD349⁻ cells are the desired cells and have viabilities greater than90%, or greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

In further embodiments, the desired cells are present at between about5×10^3 desired cells/mL and about 2.5×10^8 desired cells/mL in theunprocessed host liquid. In various further embodiments, the desiredcells may be present at between about 5×10^3 desired cells/mL and about2.5×10^7 desired cells/mL; 5×10^3 desired cells/mL and about 2.5×10^6desired cells/mL, 5×10^3 desired cells/mL and about 2.5×10^5 desiredcells/mL, 5×10^3 desired cells/mL and about 10^5 desired cells/mL, andabout 5×10^3 desired cells/mL and about 5×10^4 desired cells/mL in theunprocessed host liquid.

For example, when CD34+ cells are the desired cells and cord blood isthe host liquid, the desired cells may be present at between about5×10^3 desired cells/mL and about 5×10^4 desired cells/mL in theunprocessed cord blood, or at about 5×10^3 desired cells/mL in theunprocessed cord blood.

In a further non-limiting example, CD3+ cells are the desired cells anda leukapheresis product is the host liquid, the desired cells may bepresent at between about 2.5×10^7 desired cells/mL and about 2.5×10^8desired cells/mL. In another non-limiting example, CD3+ cells are thedesired cells and are present between about 3×10^6/mL and about6×10^6/ml in the unprocessed host liquid (whole blood).

In certain embodiments, the total cell processing time employing themethods of the invention is less than one hour, less than 45 minutes,less than 30 minutes, less than 25 minutes, less than 20 minutes, lessthan 15 minutes, less than 10 minutes, or less than 5 minutes.

In certain embodiments, appropriate containers of the functionallyclosed system (such as the processing container, the second container,and/or the transfer container) are purged with a gas mixture containingsufficient O₂ and CO₂ to meet the metabolic needs of the cells, thebalance of the gas mixture being the same perfluorocarbon gas containedin the buoyant agents (such as microbubbles).

In certain embodiments, the host liquid in which the target cells aresuspended is equilibrated with a gas mixture containing sufficient O₂and CO₂ to meet the metabolic needs of the cells, the balance of the gasmixture being the same perfluorocarbon gas contained in the in thebuoyant agents (such as gas-filled bubbles). It is well within the levelof those of skill in the art to determine suitable conditions to beemployed in carrying out the methods of the invention based on thedisclosure herein.

In another aspect, the invention provides cell suspensions comprising

(a) a liquid medium having a volume of at least 10 mL (or,alternatively, at least 25 mL, at least 50 mL, at least 75 mL, at least100 mL, at least 200 mL, at least 400 mL, at least 800 mL, etc.); and

(b) desired cells suspended in the liquid medium, wherein the desiredcells are selected from the group consisting of hematopoietic stem andprogenitor cells, mesenchymal stem and progenitor cells, endothelialprogenitor cells found in normal blood, placental/cord blood, bonemarrow, white blood cells, granulocytes, mononuclear cells, lymphocytes,monocytes, T-cells, B-cells, NK cells, the stromal vascular fractioncells resident in adipose tissue, and sub-populations of such desiredcells, wherein the desired cells are present in a liquid medium andwherein the desired cells make up at least 80% (or alternatively, atleast 85%, 90%, 95%, 98%, 99%, or more) of cells in the cell suspension;

wherein the desired cells viability is greater than 90%, or greater than95%, or greater than 97%, or greater than 99%; and

wherein the cell suspension is present within a closed cell separationsystem.

The cell suspensions of the invention are those desired cells obtainedafter sequestration (and optionally after cell detachment), but stilleither (a) contained within functionally closed system (such as the cellseparation system), or (b) obtained directly from the functionallyclosed system prior to any downstream processing that might be carriedout. The liquid medium is the liquid medium in which the cells aresuspended after sequestration/cell detachment, and thus may be any suchliquid medium suitable for a particular intended use. In variousembodiments, the liquid medium may comprise saline, cell culture medium,or any other liquid medium suitable for a particular use. In oneembodiment, the cell suspension is directly obtained from the closedcell separation system. In another embodiment, the cell separationsystem comprises the cell separation system/apparatus of any embodimentor combination of embodiments of the present invention. In a furtherembodiment, the cell suspension is present in a cell suspension removalstream via the transfer container of the closed cell separation system.In one embodiment, the desired cells in the composition comprise abuoyant label attached to the cells.

In one embodiment, the number of viable desired cells is at least 1×10³,or at least 1×10⁴, or at least 2×10⁴, or at least 5×10⁴, or at least1×10⁵, or at least, 2×10⁵, or at least 5×10⁵, or at least 1×10⁶, or atleast 2×10⁶, or at least 5×10⁶, or at least 1×10⁷, or at least 2×10⁷, orat least 5×10⁷, or at least 1×10⁸, or at least 2×10⁸, or at least 5×10⁸,or at least 1×10⁹, or at least 2×10⁹, or at least 5×10⁹. In a furtherembodiment, the desired cells comprise buoyant labels attached to thecells. In a further embodiment, the cell suspension is present within atransfer container, a processing container and/or a third container ofthe cell separation system as recited in any embodiment or combinationof embodiments disclosed herein.

In another aspect, a kit is provided comprising one or more bindingagents, and/or one or more buoyant labels, and/or one or more buoyantreagents, and one or more cartridges and/or more one or more systemsand/or one or more buffers.

In another aspect, the invention provides compositions comprising:

(a) at least one binding agent covalently or non-covalently linked to atleast one first linker, the at least one binding agent able to bind toat least one molecular target on the cells in a cell suspension; and

(b) at least one buoyant label covalently or non-covalently linked to atleast one second complementary linker, the at least one buoyant labelexhibiting a density substantially different from the density of theliquid medium in which the cells to be separated are suspended.

In another embodiment, wherein the at least one buoyant label includes,but is not limited to, a gas-filled bubble, a hollow polymer, a glassbead, a microporous bead with entrained gas, a droplet of an immiscibleliquid, a solid of any shape, a liquid of any shape, a gold nanoparticleor a silver nanoparticle. In a further embodiment, the desired cellscomprise various cell types, including but not limited to, hematopoieticstem and progenitor cells, mesenchymal stem and progenitor cells andendothelial progenitor cells found in normal blood, placental/cordblood, bone marrow or the stromal vascular fraction cells resident inadipose tissue, cultured cells or genetically modified cells.

In one embodiment, the compositions comprise

(a) at least one binding agent covalently or non-covalently linked to atleast one first linker, the at least one binding agent able to bind toat least one molecular target on the cells in a cell suspension; and

(b) at least one buoyant label covalently or non-covalently linked to atleast one second complementary linker, the at least one buoyant labelexhibiting a density substantially different from the density of theliquid medium in which the cells to be separated are suspended.

In another embodiment, the compositions comprise:

(i) a primary binding agent comprising an agent capable of binding to atleast one cellular epitope on a target cell;

(ii) a first linker bound to the agent, wherein the first linkercomprises a first oligonucleotide having a first complementary region;

(iii) a buoyant label;

(iv) a second linker bound to the buoyant label, wherein the secondlinker comprises a second oligonucleotide having a second complementaryregion perfectly complementary to the first complementary region,wherein a hybrid of the first and second linkers' complementary regionshas a calculated Tm of at least 40° C.;

wherein the first linker and the second linker are hybridized to eachother. In one embodiment, the composition further comprises a targetcell bound to the primary binding agent.

In another aspect, the invention provides kits comprising

(i) a primary binding agent comprising an agent capable of binding to atleast one cellular epitope on a target cell;

(ii) a first linker bound to the agent, wherein the first linkercomprises a first oligonucleotide having a first complementary region;

(iii) a buoyant label;

(iv) a second linker bound to the buoyant label, wherein the secondlinker comprises a second oligonucleotide having a second complementaryregion, wherein the second complementary region is perfectlycomplementarity to the first complementary region, and wherein a hybridof the first and second complementary regions has a calculated Tm of atleast 40° C.

These compositions and kits are useful, for example, in specificembodiments of the methods described above, where target cell detachmentis carried out by dehybridization. In one embodiment the calculated Tmof a hybrid of the first and second complementary region is between 40°C. and about 60° C. In another embodiment, the calculated Tm iscalculated using the nearest-neighbor two-state model:

${{Tm}\left( {{^\circ}\; C} \right)} = {\frac{\Delta\; H\;{^\circ}}{{\Delta\; S\;{^\circ}} + {R\;{\ln\lbrack{oligo}\rbrack}}} - 273.15}$where ΔH° (enthalpy) and ΔS° (entropy) are the melting parameterscalculated from the sequence and the published nearest neighborthermodynamic parameters under the ionic conditions used, R is the idealgas constant (1.987 calK⁻¹mole⁻¹), [oligo] is the molar concentration ofan oligonucleotide, and the constant of −273.15 converts temperaturefrom Kelvin to degrees of Celsius. In a further embodiment, one of thefirst linker and the second linker further comprises biotin (optionallylinked to a first member of an oligonucleotide hybridizing pair), andthe other further comprises streptavidin (optionally linked to a secondmember of an oligonucleotide hybridizing pair). In various embodiments,the buoyant labels are selected from the group consisting of gas-filledbubbles, hollow polymers, glass beads, microporous based with entrainedgas, droplets of an immiscible liquid, gold nanoparticles, and silvernanoparticles. In one specific embodiment, the buoyant labels comprisegas-filled bubbles. In another embodiment, the gas-filled bubblescomprise perfluorocarbon gas cores encompassed by lipid, phospholipid,protein or carbohydrate shells. In a further specific embodiment, thegas-filled bubbles comprise perfluorocarbon gas cores encompassed byphospholipid shells. In a further specific embodiment, the gas-filledbubbles have a diameter between about 1 um and about 6.5 um.

In one embodiment, the at least one binding agent includes, but is notlimited to, antibodies, oligonucleotides, aptamers, molecularlyimprinted polymers, carbohydrates, proteins, peptides, enzymes, smallmolecules, lipids, fatty acids, metal atoms, metal ions or syntheticpolymers. In another embodiment, the at least one first linker and/orthe second linker includes, but is not limited to, biotin, avidin,streptavidin, an oligonucleotide, an antibody-binding protein, a moietybound by an antibody-binding protein, combinations thereof, or anysecond attached binding agent. In a specific embodiment, the primarybinding agent comprises an antibody.

In another aspect, the invention provides compositions comprisingdesired cells purified via buoyancy-activated cell sorting from astarting admixture of at least one molecular type of desired cell and atleast one molecular type of non-desired cell where said startingadmixture contains at least 1 times the number of non-desired cells asdesired cells, or at least 5 times the number of non-desired cells asdesired cells, or at least 10 times the number of non-desired cells asdesired cells, or at least 50 times the number of non-desired cells asdesired cells, or at least 100 times the number of non-desired cells asdesired cells, or at least 500 times the number of non-desired cells asdesired cells, or at least 1000 times the number of non-desired cells asdesired cells wherein:

the recovery efficiency of the at least one type of desired cell isgreater than 80%, or greater than 85%, or greater than 90%, or greaterthan 95%; the purity of the at least one type of desired cell is greaterthan 80%, or greater than 85%, or greater than 90%, or greater than 95%;the viability of the at least one type of desired cell is greater than90%, or greater than 95%, or greater than 97%, or greater than 99%; and

the volume of the admixture of at least one molecular type of desiredcell and at least one molecular type of non-desired cell subjected tobuoyancy-activated cell sorting is greater than 2 mL, or greater than 10mL, or greater than 50 mL, or greater than 100 mL, or greater than 150mL, or greater than 200 mL, or greater than 400 mL, or greater than 800mL.

In various embodiments, the purified desired cells, or cells expandedfrom the purified desired cells, are used for cell therapy, research, ordrug discovery. In another embodiment, the desired cells are enrichedvia centrifugation prior to purification via buoyancy-activated cellsorting. In a further embodiment, the buoyancy-activated cell sortingstep is performed in the same container in which the preceding desiredcell enrichment was performed.

In various embodiments, the desired cells are expanded prior topurification, or subsequent to purification. In other embodiments, thecells are used for cell therapy, and the cell therapy is autologous orallogeneic. In further embodiments, the desired cells are stem orprecursor cells, immune cells, differentiated from stem cells,differentiated from induced pluripotent stem cells, geneticallyengineered cells, and/or cells to be genetically engineered.

In various further embodiments, the desired cells are negativelyselected via buoyancy-activated cell sorting, are positively selectedvia buoyancy-activated cell sorting, or a combination thereof. Forexample, the desired cells may be selected via buoyancy-activated cellsorting via a combination of sequential negative and positiveselections, wherein the sequential negative and positive selections areperformed in the same container.

Example 1

Isolation of Target Cells Using a Oligonucleotide Hybrid Linker

In order to prepare an enriched cell suspension-binding agent-buoyantlabel complex for use in the method of the present invention, thefollowing protocol may be used: A peripheral blood mononuclear cell(PBMC) preparation was prepared by processing 200 ml of fresh humanperipheral blood and a collected unit of placental cord blood. The PBMCpreparation was diluted to a WBC concentration of 1×10⁷/ml with PBSHE.The diluted PBMC preparation was enumerated using a Hematology Analyzer.

For CD3 Cell Isolation from Peripheral Blood PBMCs:

CD3 antibody and oligonucleotide hybrid was prepared by incubating CD3antibody at 0.3 mg/mL (2 μM) concentration, previously conjugated at the5′ end to 20-Mer oligo 5′-GGA AGC GGT GCT ATC CAT CT-3′ (SEQ ID NO:1)with 1/10^(th) volume complementary oligonucleotide (A′) (4 μmconcentration) of either 8-Mer, 10-Mer, 12-Mer or 14-Mer previouslyconjugated to biotin per lmL cell suspension. The complementary oligosequence is the 3′-most bases of the 20-Mer oligo. Oligonucleotides wereincubated at room temperature for one hour for hybridization to occur.

To the diluted PBMC preparation, 12.5 μg CD3 antibody-oligo hybrid wasadded per mL cell suspension (previously determined experimentally to beoptimal for CD3+ cell selection) and incubated on a rotating mixer atroom temperature for 20 minutes.

For CD34 Cell Isolation from Cord Blood PBMCs:

CD34 antibody and oligonucleotide hybrid was prepared by incubating CD34antibody at 0.1 mg/mL (2 μM) concentration, previously conjugated at the5′ end to 20-Mer oligo 5′-GGA AGC GGT GCT ATC CAT CT-3′ (SEQ ID NO:1)with 1/10^(th) volume complementary oligonucleotide (A′) (4 μmconcentration) of either 8-Mer, 10-Mer, 12-Mer or 14-Mer previouslyconjugated to biotin per 1 mL cell suspension. The complementary oligosequence is the 3′-most bases of the 20-Mer oligo. Oligonucleotides wereincubated at room temperature for one hour for hybridization to occur.

To the diluted PBMC preparation, 15 μg CD34 antibody-oligo hybrid wasadded per mL cell suspension (previously determined experimentally to beoptimal for CD34+ cell selection) and incubated on a rotating mixer atroom temperature for 20 minutes.

Separately, a buoyant reagent was prepared by briefly vortexing a vialof Advanced Microbubbles SIMB™4-5 to re-suspend the microbubbles; theconcentration of the microbubble suspension was determined by analyzinga 2 μl aliquot on a MultiSizer™ 4 (Beckman Coulter). A ratio of 10microbubbles per WBC was used to calculate the volume of microbubbles tobe added to the PBMC suspension for CD3 isolation and a ratio of 50microbubbles per WBC was used to calculate the volume of microbubbles tobe added to the PBMC suspension for CD34 isolation. ThePBMC-antibody-oligo hybrid-microbubble suspension was incubated on arocking table at room temperature for 40 minutes.

Following incubation, the cell suspension was centrifuged at 400×g for 5minutes. The microbubbles (and microbubble-bound cells) formed a cakefloating on the meniscus (positive fraction), while unbound cells formeda pellet at the bottom of a container (negative fraction), and a clearsolution was observed between the cell pellet and the bubble cake.

The positive fraction was transferred to a separate container to which a30-fold excess of oligo A′ was added and incubated in a waterbath at 37°C. for 20 minutes and then to a rotating mixer in a 37° C. incubator for30 minutes to dehybridize the oligonucleotide. The microbubble-positivecell fraction was centrifuged at 400×g for 5 minutes. The now unboundtarget cell population formed a pellet at the bottom of the containerwhereas microbubbles formed cake floating on the meniscus. Microbubbleswere removed from the container.

Diluted PBMC, negative fraction, positive fraction and microbubblesamples were subjected to flow cytometric analysis. An antibodymaster-mix was used, containing FITC-labeled anti-human CD45,7-aminoactinoamycin dye (7-AAD), and APC-labeled anti-human CD3 for CD3isolation or PE-labeled anti-human CD34 for CD34 isolation. 50 μlaliquots of each cell suspension were added to flow tubes containing avolume of master-mix previously determined to be suitable. 50 μl ofdiluted PBMC suspension was added to both a positive control and anegative control flow tube. The positive control was labeled withmaster-mix, and the negative control tube was labeled with FITC-labeledanti-human CD45, 7-AAD, and PE or APC-labeled mouse IgG (isotype), Alltubes were then vortexed and incubated for 20 minutes at roomtemperature in the dark. After this incubation, all tubes received 0.5ml of 1× lysis buffer, then the tubes were vortexed and incubated atroom temperature in the dark for an additional 10 minutes. Immediatelypreceding flow cytometric analysis each tube received 50 μl of flowcount bead suspension. The tubes were then analyzed using aCD45/7-AAD/CD3 gating strategy previously determined to yield adequateresults with PBMCs for CD3 isolation or ISHAGE gating strategy for CD34isolation.

Experimental results are displayed in FIG. 11. 8-Mer (n=1) and 10-Mer(n=1) and 12-Mer (n=1) oligonucleotide linkers with calculated Tm of16.3° C., 27.9° C. and 41.6° C. yielded target cell recovery of 12.5%and 21.7% and 68.4% respectively. 14-Mer (n=3) oligonucleotide linkerswith calculated Tm of 50.2° C. yielded target cell recovery between76.7% and 94.5%. These results demonstrate that target cell recovery isa sensitive function of oligonucleotide linker length, and provide thesurprising finding that highest recovery is achieved witholigonucleotide linker hybrids having a calculated Tm (under the ionicconditions employed) that is substantially higher than thedehybridization temperature of 37° C. (which is ideal for human cellviability) employed here. Under optimal conditions target cellrecoveries of up to 95% were observed, which those skilled in the artwill recognize exceed the recovery efficiency of conventional targetcell isolation methods.

Example 2

Isolation of Target Cells from Human Blood in a Functionally ClosedSystem

In order to prepare an enriched cell suspension-binding agent-buoyantlabel complex for use in the method of the present invention, thefollowing protocol was used: Peripheral blood and leukapheresismononuclear cell (PBMC) preparations were prepared by processing 200 mlof fresh human peripheral blood for CD3+ or CD4+ isolation and 100 mLleukapheresis for CD3+ isolation in the present invention and set toharvest 40-45 mL into PBSHE. After evacuation of the non-target cellfractions within the cartridges, the target cell enriched cellsuspensions were transferred to the processing containers by means ofthe transfer container to maintain sterility.

For CD3+ Cell Isolation from Peripheral Blood:

PBMCs or leukapheresis PBMCs, 1 mL Biotinylated CD3 antibody (stockconcentration 0.5 mg protein/mL) was diluted to a volume of 5 mL withPBSHE and added through an integrated 0.2 μm filter and incubated withconstant mixing at room temperature for 20 minutes.

For CD4+ Cells from Peripheral Blood:

1 mL Biotinylated CD3 antibody (stock concentration 0.5 mg protein/mL)was diluted to a volume of 5 mL with PBSHE and added through anintegrated 0.2 μm filter and incubated with constant mixing at roomtemperature for 20 minutes.

CD4+ Cells

0.5 mL Biotinylated CD3 antibody (stock concentration 0.5 mg protein/mL)was diluted to a volume of 5 mL with PBSHE and added through anintegrated 0.2 μm filter and incubated with constant mixing at roomtemperature for 20 minutes.

Separately, a buoyant reagent was prepared by briefly vortexing 2×1.4 mLvials of Advanced Microbubbles SIMB™4-5 microbubbles for CD3+ or CD4+and aseptically dispensing into a 5 mL syringe with a PVC output tube.The remainder of the 5 mL made up with PBSHE. Following completion ofthe 20 minute antibody incubation, the microbubble syringe was sterilewelded to the 3rd conduit of the system and microbubbles added. ThePBMC-antibody-microbubble suspension was incubated with constant mixingat room temperature for 30 minutes.

Following incubation, the system was centrifuged at 400×g for 5 minutes.The microbubbles (and microbubble-bound cells) formed a cake floating onthe meniscus, while unbound cells formed a pellet at the bottom of acontainer, and a clear solution was observed between the cell pellet andthe bubble cake. The system was then centrifuged at 50×g for 1 minute totransfer the negative fraction to the second container, in this caseserving as the depletion container. An aliquot of the negative was takenfor cell enumeration

The bubble cakes plus supernatant was transferred to the transfercontainer and then subjected to ultrasonic energy for 2 seconds, untilbubbles were disrupted. The sonicated, positive cell fraction wastransferred back to the processing container and centrifuged at 400×gfor 5 minutes then transferred to the third container, in this caseserving as the harvest container to a defined final volume. An aliquotof the positive fraction was taken for cell enumeration.

Diluted PBMC, negative fraction and positive fraction samples weresubjected to flow cytometric analysis. An antibody master-mix was used,containing FITC-labeled anti-human CD45, 7-aminoactinoamycin dye(7-AAD), PE-labeled anti-human CD4 and APC-labeled anti-human CD3. 50 μlaliquots of each cell suspension were added to flow tubes containing avolume of master-mix previously determined to be suitable. 50 μl ofdiluted PBMC suspension was added to both a positive control and anegative control flow tube. The positive control was labeled withmaster-mix, and the negative control tube was labeled with FITC-labeledanti-human CD45, 7-AAD, and PE or APC-labeled mouse IgG (isotype), Alltubes were then vortexed and incubated for 20 minutes at roomtemperature in the dark. After this incubation, all tubes received 0.5ml of 1× lysis buffer, then the tubes were vortexed and incubated atroom temperature in the dark for an additional 10 minutes. Immediatelypreceding flow cytometric analysis each tube received 50 μl of flowcount bead suspension. The tubes were then analyzed using aCD45/CD4/7-AAD/CD3 gating strategy previously determined to yieldadequate results with PBMCs.

Experimental results are shown in FIG. 12. Isolation of cells fromperipheral blood yielded recovery of CD3+ (n=4) between 80.9% and 88.1%,purity between 94.8% and 99.6% and viability between 90.7% and 95.8%.For CD4+ cells (n=1) recovery was 81.7%, purity 96.6% and viability95.3%. Isolation of CD3+ cells from leukapheresis (n=1) yielded recoveryof 83.0%, purity 98.6% and viability 96.1%. In each case, these combinedrecovery, purity, and viability metrics exceed those previously reportedin the literature by workers employing entirely manual processes andopen containers, demonstrating the improved performance of the presentinvention.

Example 3

Isolation of CD3+ Cells from Human Blood in a Functionally Closed SystemUsing Oligonucleotide Linkers

In order to prepare an enriched cell suspension-binding agent-buoyantlabel complex for use in the method of the present invention, thefollowing protocol may be used: A peripheral blood mononuclear cell(PBMC) preparation was prepared by processing 200 ml of fresh humanperipheral blood in the present invention and set to harvest 40-45 mLinto PBSHE. After evacuation of the non-target cell blood fractions fromthe cartridge, the harvested enriched target cell suspension wastransferred to the processing container via the transfer container tomaintain sterility.

CD3 antibody and oligonucleotide hybrid was prepared by incubating 540μg CD3 antibody at 3 mg/mL (2 μM) concentration, previously conjugatedat the 5′ end to 20-Mer oligo 5′-GGA AGC GGT GCT ATC CAT CT-3′ (SEQ IDNO:1) with 1/10^(th) volume complementary oligonucleotide (A′) (4 μmconcentration) of 14-Mer previously conjugated to biotin. Thecomplementary oligo sequence is the 3′-most bases of the 20-Mer oligo.Oligonucleotides were incubated at room temperature for one hour forhybridization to occur. Following hybridization antibody-oligo hybridwas added to PBMC through an integrated 0.2 μm filter and incubated withconstant mixing at room temperature for 20 minutes.

Separately, a buoyant reagent was prepared by briefly vortexing 2×1.4 mlvials of Advanced Microbubbles SIMB™4-5 microbubbles and asepticallydispensing into a 5 mL syringe with a PVC output tube. The remainder ofthe 5 mL made up with PBSHE. Following completion of the 20 minuteantibody incubation, the microbubble syringe was sterile welded to the3rd conduit of the system and microbubbles added. ThePBMC-antibody-microbubble suspension was incubated with constant mixingat room temperature for 30 minutes.

Following incubation, the system was centrifuged at 400×g for 5 minutes.The microbubbles (and microbubble-bound cells) formed a cake floating onthe meniscus, while unbound cells formed a pellet at the bottom of acontainer, and a clear solution was observed between the cell pellet andthe bubble cake. The system was then centrifuged at 50×g for 1 minute totransfer the negative fraction to the second container, the depletioncontainer. An aliquot of the negative was taken for cell enumeration.Separately, a 600 μM concentration of competing Oligo-A′ was combined ina 5 mL syringe with PBSHE.

The bubble cakes plus supernatant were transferred to the transfercontainer and the competing Oligo-A′ was aseptically added. ThePBMC-antibody-microbubble suspension was incubated with competing OligoA′ in a waterbath at 37° C. with constant mixing for 40 minutes.

The positive cell fraction was transferred back to the processingcontainer and centrifuged at 400×g for 5 minutes then transferred to thethird container, in this case serving as the harvest container, to adefined final volume. The unbound microbubbles remained in theprocessing container. An aliquot of the positive and microbubblefractions was taken for cell enumeration.

Diluted PBMC, negative fraction, positive and microbubble fractionsamples were subjected to flow cytometric analysis. An antibodymaster-mix was used, containing FITC-labeled anti-human CD45,7-aminoactinoamycin dye (7-AAD), and APC-labeled anti-human CD3. 50 μlaliquots of each cell suspension were added to flow tubes containing avolume of master-mix previously determined to be suitable. 50 μl ofdiluted PBMC suspension was added to both a positive control and anegative control flow tube. The positive control was labeled withmaster-mix, and the negative control tube was labeled with FITC-labeledanti-human CD45, 7-AAD, and APC-labeled mouse IgG (isotype), All tubeswere then vortexed and incubated for 20 minutes at room temperature inthe dark. After this incubation, all tubes received 0.5 ml of 1× lysisbuffer, then the tubes were vortexed and incubated at room temperaturein the dark for an additional 10 minutes. Immediately preceding flowcytometric analysis each tube received 50 μl of flow count beadsuspension. The tubes were then analyzed using a CD45/7-AAD/CD3 gatingstrategy previously determined to yield adequate results with PBMCs.

Experimental Results are Shown in FIG. 11.

Isolation of CD3+ cells from peripheral blood (n=1) using a 14-Meroligonucleotide linker with a calculated Tm of 50.2° C. (n=1) yieldedrecovery of 76.7%, purity 97.3% and viability 96.8%. These resultsdemonstrate that the oligonucleotide linker method yields excellentpurity, recovery, and viability in an automated large-volume systemwhile obviating the need for physical disruption of microbubbles (suchas via sonication) in order to collect target cells, which could bedifficult to control precisely in large-volume systems such as thatemployed here.

We claim:
 1. A cell separation system, comprising: (a) a cartridge comprising (i) a processing container comprising at least one input port, a first exit port, and a second exit port; (ii) a second container comprising an input port; (iii) a third container comprising an input port and a first exit port; (iv) a first conduit connecting the first exit port of the processing container and the input port of the second container, wherein the first conduit comprises a first reversible closing device, wherein the second container is transiently fluidically connected to the processing container such that fluid flow only from the processing container to the second container may occur when the first reversible closing device is opened; (v) a second conduit connecting the second exit port of the processing container and the input port of the third container, wherein the second conduit comprises a second reversible closing device, wherein the third container is transiently fluidically connected to the processing container such that fluid flow only from the processing container to the third container may occur when the second reversible closing device is opened; (b) a transfer container transiently fluidically connected to a T or Y connector, wherein the T or Y connector includes a first input port of the transfer container and an exit port of the transfer container; (c) a third conduit connecting the exit port of the third container to the first input port of the transfer container, wherein the third conduit comprises a third reversible closing device, such that the third container is transiently fluidically connected to the transfer container such that fluid flow only from the third container to the transfer container may occur when the third reversible closing device is opened; (d) a fourth conduit connecting the exit port of the transfer container to the at least one input port of the processing container, wherein the fourth conduit comprises a fourth reversible closing device, such that the transfer container is transiently fluidically connected to the processing container, such that fluid flow only from the transfer container to the processing container may occur when the fourth reversible closing device is opened; and (e) a control module configured to control the first reversible closing device and/or the second reversible closing device.
 2. The cell separation system of claim 1, wherein the T or Y connector is disposed between the third container and the transfer container and between the transfer container and the processing container.
 3. The cell separation system of claim 1, wherein the at least one input port of the processing container comprises a first input port and a second input port, wherein the fourth conduit connects the exit port of the transfer container to the first input port of the processing container.
 4. The cell separation system of claim 3, further comprising a first medium input conduit connecting the second input port of the processing container to at least one medium reservoir, wherein the first medium input conduit comprises at least a fifth reversible closing device, wherein the at least one medium reservoir is transiently fluidically connected to the processing container such that fluid flow only from the at least one medium reservoir to the processing container may occur when the at least fifth reversible closing device is opened.
 5. The cell separation system of claim 3, wherein the at least one input port of the processing container further comprises a third input port coupled to a fifth conduit.
 6. The cell separation system of claim 1, wherein the transfer container is transiently fluidically connected to a second input port.
 7. The cell separation system of claim 6, further comprising a second medium input conduit connecting the second input port of the transfer container to at least one medium reservoir, wherein the second medium input conduit comprises at least a sixth reversible closing device, wherein the at least one medium reservoir is transiently fluidically connected to the processing container such that fluid flow only from the at least one medium reservoir to the transfer container may occur when the at least sixth reversible closing device is opened.
 8. The cell separation system of claim 7, wherein the first medium input conduit and/or the second medium input conduit further comprise a filter.
 9. The cell separation system of claim 1, wherein the cell separation system further comprises a mixer.
 10. The cell separation system of claim 9, wherein the mixer comprises a static mixer.
 11. The cell separation system of claim 9, wherein the mixer comprises an impeller disposed on an internal surface of a roof of the cartridge.
 12. The cell separation system of claim 9, wherein the mixer comprises an impeller spaced away from an internal surface of a roof of the cartridge.
 13. The cell separation system of claim 9, wherein the mixer comprises a peristaltic pump comprising a pump conduit having a first end and a second end, wherein the first end of the pump conduit is positioned in the processing chamber, and wherein the second end of the pump conduit is positioned outside of the processing chamber and is connected to the at least one input port of the processing chamber, wherein the peristaltic pump is configured to compress a portion of the pump conduit such that that the portion of the pump conduit under compression is pinched closed thus forcing fluid to be pumped to move through the pump conduit as the peristaltic pump rotates.
 14. The cell separation system of claim 9, wherein the mixer comprises a mixing module comprising a bottom portion and a top portion, wherein the cartridge is configured to be positioned in the bottom portion, and wherein the top portion is configured to be removably coupled to the bottom portion.
 15. The cell separation system of claim 14, wherein the mixing module includes a drive shaft coupled to the bottom portion, and wherein the drive shaft is configured to rotate the cartridge on its vertical axis by 180 degrees or by 360 degrees.
 16. The cell separation system of claim 15, further comprising a motor coupled to the drive shaft, wherein a rotation of the motor translates to a rotation of the bottom portion of the mixing module.
 17. The cell separation system of claim 14, wherein the mixing module is configured to increase a temperature of the cartridge when the cartridge is positioned in the bottom portion of the mixing module.
 18. The cell separation system of claim 17, wherein the increase in temperature of the cartridge occurs through conduction, convection, or radiation heating in the bottom portion of the mixing module.
 19. The cell separation system of claim 14, wherein the bottom portion of the mixing module is configured to vibrate to assist in mixing.
 20. The cell separation system of claim 14, further comprising a mixing control module including a control panel and a display, wherein the control panel is configured to receive one or more user inputs including a time period for mixing and one or more mixing parameters.
 21. The cell separation system of claim 20, wherein the one or more mixing parameters include 180 degree mixing, 360 degree mixing, heating the bottom portion of the mixing module at a specific temperature, and/or vibrating the bottom portion of the mixing module.
 22. The cell separation system of claim 1, wherein the second container comprises an exit port coupled to a first waste conduit.
 23. The cell separation system of claim 1, wherein the processing container further comprises a sterile vent coupled to a second waste conduit.
 24. The cell separation system of claim 1, wherein the transfer container is a rigid container.
 25. The cell separation system of claim 24, wherein the rigid container comprises polycarbonate.
 26. The cell separation system of claim 1, wherein the transfer container is a flexible container.
 27. The cell separation system of claim 26, wherein the flexible container comprises a bag. 